Preparation method for adiposome

Abstract

Provided is a preparation method for an adiposome, comprising the following steps: S1) subjecting polar lipids and neutral lipids to intermittent ultrasonic emulsification in a buffer solution to obtain a crude emulsion; and S2) purifying the crude emulsion to obtain the adiposome. Compared with the prior art, the present invention uses an ultrasonic emulsification method to prepare the adiposome, so that the quality of the adiposome prepared in different batches is controllable, and the preparation of the adiposome can be efficiently completed. Moreover, high-energy mechanical vibration and ultrasonic cavitation effects of ultrasonic waves are utilized in the ultrasonic emulsification method to widen the selection of raw materials for adiposomes, so that the method can be used to not only prepare an adiposome with unsaturated fatty acid chain phospholipids and neutral lipids as raw materials, but also synthesize an adiposome with saturated fatty acid chain phospholipids, neutral lipids, or high-melting-point lipids as raw materials. In addition, the utilization of raw materials can be improved, and the scale of adiposome production can be expanded.

Description

A method for preparing fat bodies

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on March 5, 2024, with application number 202410251149.0 and invention name “A Method for Preparing Fat Bodies”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biomedical technology, and in particular relates to a large-scale preparation method of fat bodies. Background Art

[0003] Fat bodies are artificially synthesized nanospheres that mimic the lipid structure of lipoproteins or lipid droplets in cells. The main structural feature of fat bodies is a single-molecule phospholipid layer wrapped around a neutral lipid core, with a hydrophobic interior and a hydrophilic outer surface. Fat bodies have significant structural differences from previously disclosed liposome nanoparticles, lipid nanoparticles for nucleic acid delivery, and exosome nanoparticles. The internal hydrophobic neutral lipid core gives fat bodies a natural advantage in delivering hydrophobic small molecules. At the same time, the specific monolayer phospholipid membrane on the periphery simulates the phospholipid membrane structure of lipid droplets or lipoproteins, which can recruit specific proteins and provide a basis for the design of targeting molecules.

[0004] Nanoemulsions are prepared using methods such as high-pressure homogenization, microfluidics, ultrasound, phase inversion emulsification, and self-emulsification. The basic principle of high-pressure homogenization is to generate high pressures of up to 300 MPa in the chamber during the delivery stroke, forcing a coarse emulsion through the micron-sized orifices of the homogenizer valve. Turbulence, shear stress, and cavitation transform the coarse emulsion into finer droplets. The high-pressure homogenization emulsification process consists of two main stages: first, the dispersed phase is disrupted, resulting in the formation of tiny droplets in the homogenization chamber, increasing the surface area. Then, emulsifier molecules accumulate at the newly formed interface, stabilizing the droplets. Repeated high-pressure homogenization can generate a large number of nanodroplets. The microfluidics method uses microchannels with a size range of 50 to 300 μm to generate nanodroplets. Its basic principle is similar to that of high-pressure homogenization. The crude emulsion is passed through the microchannel at a high pressure and high flow rate at the inlet, and is divided into two branches through the branch channel. Then, the branches are reconnected in the downstream interactive channel. In the interaction chamber, the crude emulsions from the two channels collide with each other at a very high speed, and the shear rate can reach 107 s. -1, thereby breaking up the coarse emulsion into a fine emulsion. Ultrasonic methods utilize the mechanical vibrations and acoustic cavitation generated by ultrasound to prepare nanoemulsions. The intense shock waves produced by ultrasound and cavitation create high pressure and turbulence, causing the microdroplets to collapse and break up, producing fine nanoemulsions. However, ultrasonic treatment requires an optimal level of input energy to achieve the smallest droplet diameter. Excessively high or low energy levels result in poor emulsification. Ultrasonic probes are often used to emulsify droplets to produce nanoemulsions, and benchtop sonicators are also used for small-scale nanoemulsion production. These are commonly used high-energy input methods for preparing nanoemulsions. Several low-energy input methods are also used to prepare nanoemulsions. Phase transfer emulsification exploits the fact that some emulsifiers, at a fixed composition, change their hydrophilicity or lipophilicity with temperature. Some nonionic surfactants change their spontaneous curvature due to hydration of their polar ends, forming oil-in-water emulsions at low temperatures. At high temperatures, this decreases as the emulsifier's solubility in water increases, resulting in water-in-oil emulsions. The temperature at which an oil-in-water emulsion transforms into a water-in-oil emulsion is called the phase transition temperature. Emulsions can form near this temperature, but they are unstable and require rapid cooling or heating to produce kinetically stable emulsions with small particle sizes and narrow particle size distributions. Self-emulsification is another low-energy emulsion synthesis method. This method involves mixing water, oil, and an emulsifier at a specific temperature. Gentle mechanical agitation allows the emulsifier to enter the aqueous phase, increasing the oil-water interfacial area and forming nanodroplets.

[0005] However, the above-mentioned emulsion synthesis methods each have disadvantages: high-pressure homogenization and microfluidics easily form amorphous membrane structures mixed in the emulsion; phase transfer method and self-emulsification method require specific emulsifiers (surfactants), which do not conform to the biocompatibility principle of fat body raw materials. Published fat body synthesis technologies are mainly vortex and ultrasound. Their basic principle is to use mechanical force or ultrasonic cavitation to force phospholipids and triglycerides to self-assemble in an aqueous system to form a single-layer phospholipid membrane coated with triglyceride core nanospheres (patent No. CN105483076B, US10987431B2, EP3395329A). The vortex method is to introduce phospholipids and neutral lipids into an aqueous system (buffer) respectively, and high-speed vortex separation of large oil droplets into small oil droplets. At the same time, phospholipid molecules can form a single-layer phospholipid membrane on the surface of the small oil droplets. After multiple vortexes, the large droplets are homogenized into small droplets, and then separated into fat bodies through a purification step. While existing vortexing techniques can produce high-purity liposomes, their phospholipid utilization rate is very low (<10%). Furthermore, the process is complex and requires manual operation (using a vortexer for 10 seconds x 24 cycles). Therefore, this technique is suitable for preparing small batches of liposomes for laboratory research but not for large-scale production. Ultrasonic methods utilize the mechanical vibrations generated by an ultrasonic probe combined with ultrasonic cavitation and turbulence to break up large neutral lipid droplets into smaller ones. Phospholipid molecules are then assembled on the surface of the neutral lipid droplets to form liposomes. However, current ultrasonic liposome preparation requires either probe ultrasound or water-bath ultrasonic emulsification in an ultrasonic cleaning machine. The resulting liposomes also suffer from low phospholipid utilization, making them unsuitable for large-scale production.

[0006] Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing fat bodies, which has a high raw material utilization rate and can be produced on a large scale.

[0008] The present invention provides a method for preparing fat bodies, comprising the following steps:

[0009] S1) treating polar lipids and neutral lipids in a buffer solution by intermittent ultrasonic emulsification to obtain a crude emulsion;

[0010] S2) purifying the crude emulsion to obtain fat bodies.

[0011] Preferably, the amplitude of the intermittent ultrasonic emulsification treatment is 40% to 100%; the frequency of the intermittent ultrasonic emulsification treatment is 20 to 50 kHz; the temperature of the intermittent ultrasonic emulsification treatment is 25° C. to 35° C.; and the time of the intermittent ultrasonic emulsification treatment is 5 to 20 minutes.

[0012] Preferably, the intermittent phacoemulsification treatment is specifically as follows:

[0013] The ultrasonic emulsification treatment is performed for 1 to 5 minutes, and then stopped for 20 to 60 seconds, and this cycle is repeated until the intermittent ultrasonic emulsification treatment time is 5 to 20 minutes.

[0014] Preferably, the step S1) is specifically as follows:

[0015] Polar lipids and neutral lipids are directly mixed, and then a buffer solution is added and subjected to intermittent ultrasonic emulsification treatment to obtain a coarse emulsion.

[0016] Preferably, the step S1) is specifically as follows:

[0017] After mixing an organic solution containing polar lipids with an organic solution containing neutral lipids, removing the solvent, adding a buffer solution and performing intermittent ultrasonic emulsification treatment, a coarse emulsion is obtained.

[0018] Preferably, the step S1) is specifically as follows:

[0019] Neutral lipids are added to a buffer solution containing polar lipids and subjected to intermittent ultrasonic emulsification to obtain a coarse emulsion.

[0020] Preferably, the step S1) is specifically as follows:

[0021] Part of the neutral lipids is added to a buffer solution containing polar lipids and then subjected to intermittent ultrasonic emulsification treatment, and the remaining neutral lipids are added in batches after one or more intervals in the intermittent ultrasonic emulsification process to obtain a coarse emulsion.

[0022] Preferably, the step S1) is specifically as follows:

[0023] An alcohol solution containing polar lipids is added to a buffer solution containing neutral lipids and subjected to intermittent ultrasonic emulsification treatment to obtain a coarse emulsion.

[0024] Preferably, the step S1) is specifically as follows:

[0025] A portion of the alcohol solution containing polar lipids is added to a buffer solution containing neutral lipids and then subjected to intermittent ultrasonic emulsification treatment, and the remaining alcohol solution containing polar lipids is added in batches after one or more intervals in the intermittent ultrasonic emulsification process to obtain a coarse emulsion.

[0026] Preferably, the step S1) is specifically as follows:

[0027] An alcohol solution containing polar lipids is added to a buffer solution containing neutral lipids and subjected to intermittent ultrasonic emulsification treatment to obtain a coarse emulsion.

[0028] Preferably, the step S1) is specifically as follows:

[0029] A portion of the alcohol solution containing polar lipids is added to a buffer solution containing neutral lipids and then subjected to intermittent ultrasonic emulsification treatment, and the remaining alcohol solution containing polar lipids is added in batches after one or more intervals in the intermittent ultrasonic emulsification process to obtain a coarse emulsion.

[0030] Preferably, the step S2) is specifically as follows:

[0031] Purifying the crude emulsion by stepwise centrifugation to obtain fat bodies;

[0032] The stepwise centrifugation specifically comprises the following steps: centrifuging the crude emulsion at 500-2000×g for 5-10 minutes to collect the lower emulsion; and centrifuging the lower emulsion at 18000-21000×g for 5-10 minutes to obtain the fat body.

[0033] Preferably, the step S2) is specifically as follows:

[0034] The crude emulsion is mixed with a density gradient centrifuge solution, and then a buffer solution is added and centrifuged stepwise to obtain fat bodies; the density gradient centrifuge solution comprises a density gradient medium and a buffer solution; the mass concentration of the density gradient medium in the density gradient centrifuge solution is 50% to 80%;

[0035] The volume of the density gradient centrifugation fluid is 5% to 20% of the volume of the crude emulsion.

[0036] Preferably, the stepwise centrifugation is specifically as follows:

[0037] First, centrifuge at 1000-1600×g for 5-10 min, then at 16000-18000×g for 5-10 min, and then at 20000-21000×g for 5-10 min. Collect fat bodies from the top of the buffer after each centrifugation.

[0038] Preferably, the step S2) specifically comprises: performing a first centrifugation on the crude emulsion in an ultrafiltration centrifuge tube, collecting the emulsion centrifuged by ultrafiltration, and performing a second centrifugation on the remaining emulsion to obtain fat bodies;

[0039] The collected emulsion obtained by ultrafiltration centrifugation is subjected to high-speed centrifugation to obtain fat bodies.

[0040] Preferably, the centrifugal force of the first centrifugation and the second centrifugation are each independently 500-1000×g;

[0041] The time of the first centrifugation and the second centrifugation are each independently 5 to 10 minutes;

[0042] The first centrifugation and the second centrifugation are both performed at 2°C to 10°C;

[0043] The high-speed centrifugation speed is 16000-20000×g;

[0044] The time of the high-speed centrifugation is 5 to 10 minutes.

[0045] Preferably, the polar lipids include one or more of phospholipids, functional polar lipids and cationic lipids;

[0046] The phospholipid is selected from one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and sphingomyelin;

[0047] The functional polar lipid is selected from one or more of polyethylene glycol-modified sterols, biotin-modified sterols, amino acid-modified sterols, polypeptide-modified sterols, polysaccharide-modified sterols, nucleic acid-modified sterols, polyethylene glycol-modified phospholipids, biotin-modified phospholipids, amino acid-modified phospholipids, polypeptide-modified phospholipids, polysaccharide-modified phospholipids and nucleic acid-modified phospholipids;

[0048] The cationic lipid is selected from one or more of (2,3-dioleoyl-propyl)-trimethylammonium-chloride, (2,3-dioleoyl-propyl)-trimethylamine, 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylamine hydrochloride, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl](nickel salt) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride;

[0049] The neutral lipids are selected from one or more of triglycerides, wax esters, sterol esters, sterol esters, retinol esters, ether esters, polyhydroxyalkanoates and fat-soluble vitamins, such as vitamins A, D, E, and K.

[0050] Preferably, the polar lipids include one or both of functional polar lipids and cationic lipids and phospholipids;

[0051] The mass ratio of the sum of the masses of one or both of the functional polar lipids and cationic lipids to the phospholipids is (0-100):(100-0), more preferably (0-80):(100-20), further preferably (0-60):(100-40), and further preferably (0-50):(100-50).

[0052] Preferably, the mass of the polar lipids is 2.5% to 25% of the mass of the neutral lipids, more preferably 2.5% to 23%, and even more preferably 2.5% to 22%.

[0053] The present invention provides a method for preparing fat bodies, comprising the following steps: S1) subjecting polar lipids and neutral lipids to intermittent ultrasonic emulsification in a buffer solution to obtain a crude emulsion; S2) purifying the crude emulsion to obtain fat bodies. Compared with existing technologies, the present invention utilizes ultrasonic emulsification, resulting in stable and controllable quality of fat body preparation between batches, allowing for efficient fat body preparation. Furthermore, ultrasonic emulsification utilizes high-energy mechanical vibration and ultrasonic cavitation to expand the range of fat body raw materials available. Fat bodies can be prepared not only from unsaturated fatty acid chain phospholipids and neutral lipids, but also from phospholipids with saturated fatty acid chains, neutral lipids, or high-melting-point lipids. Furthermore, the present method can improve raw material utilization and expand the scale of fat body production. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG1 is a schematic diagram of a specific production process of fat bodies provided by the present invention;

[0055] FIG2 is a graph showing the particle size and monodispersity of the emulsions obtained at different ultrasonic treatment times;

[0056] FIG3 is a particle size distribution diagram of fat bodies prepared at different lipid ratios in Example 1 of the present invention;

[0057] FIG4 is a diagram showing the microscopic morphology and average particle size of fat bodies collected under different centrifugation conditions in the 500 μg group in Example 1 of the present invention;

[0058] FIG5 is a microscopic morphology of fat bodies collected under different centrifugation conditions in the 500 μg group in Example 1 of the present invention observed under transmission electron microscopy;

[0059] FIG6 is a diagram showing the calculation results of lipid utilization of fat bodies in Example 1 of the present invention;

[0060] FIG7 is a diagram showing the results of a fat body stability test in Example 1 of the present invention;

[0061] FIG8 is a schematic diagram of the process for preparing fat bodies in Example 2 of the present invention;

[0062] FIG9 is a graph showing the appearance and OD600 concentration of different fat bodies obtained in Example 2 of the present invention;

[0063] FIG10 is a microscopic morphology of fat body components at different phospholipid neutral lipid ratios at 5.0% in Example 2 of the present invention;

[0064] FIG11 is a microscopic morphology of fat body components at different phospholipid neutral lipid ratios of 7.5% in Example 2 of the present invention;

[0065] FIG12 is a microscopic morphology of fat body components at different phospholipid neutral lipid ratios of 10.0% in Example 2 of the present invention;

[0066] FIG13 is a particle size distribution diagram of fat body components with different phospholipid neutral lipid ratios in Example 2 of the present invention;

[0067] FIG14 is a microscopic morphology of TOG fat bodies in Example 3 of the present invention;

[0068] FIG15 is a microscopic morphology of fish oil fat bodies in Example 3 of the present invention;

[0069] FIG16 is a particle size distribution diagram of fat body components synthesized from different polar lipids and neutral lipids in Example 4 of the present invention;

[0070] FIG17 is a particle size distribution diagram of fat body components synthesized with neutral lipids at different ratios of DOPC and cholesterol in Example 5 of the present invention;

[0071] FIG18 is a particle size distribution diagram of fat body components synthesized with neutral lipids at different ratios of DPPC and cholesterol in Example 6 of the present invention;

[0072] FIG19 is a particle size distribution diagram of fat body components synthesized from neutral lipids at different ratios of DSPC and cholesterol in Example 7 of the present invention;

[0073] FIG20 is a graph showing the particle size distribution of fat body components synthesized from different phospholipids and cholesterol oleate in Example 8 of the present invention. DETAILED DESCRIPTION

[0074] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0075] The present invention provides a method for preparing fat bodies, comprising the following steps: S1) subjecting polar lipids and neutral lipids to intermittent ultrasonic emulsification in a buffer solution to obtain a crude emulsion; S2) purifying the crude emulsion to obtain fat bodies.

[0076] The present invention has no particular limitation on the sources of all raw materials, and any commercially available raw materials may be used.

[0077] The present invention uses micro, small, medium, large, or ultra-large ultrasonic emulsifiers as the primary equipment for fat body production. Polar lipids (including one or more of phospholipids, functional polar lipids, and cationic lipids) and neutral lipids are emulsified in a buffer solution to produce a crude emulsion. This crude emulsion is then subjected to stepwise centrifugation to obtain the final fat body product. This method improves the utilization rate of fat body raw materials, enabling the rapid and efficient production of high-purity fat bodies, paving the way for large-scale fat body production.

[0078] According to the present invention, the polar lipids are polar lipids well known to those skilled in the art, and are not particularly limited. In the present invention, they preferably include but are not limited to one or more of phospholipids, functional polar lipids and cationic lipids; in some specific embodiments provided by the present invention, the polar lipids include phospholipids; in other specific embodiments provided by the present invention, the polar lipids include one or two of functional polar lipids and cationic lipids and phospholipids; the mass ratio of the sum of the mass of one or two of the functional polar lipids and cationic lipids to the mass of phospholipids is preferably (0-100): (100-0), more preferably (0-80): (100-20), and even more preferably (0-60): (100-40 ), more preferably (0-50):(100-50); in the present invention, the mass ratio of the sum of the mass of one or both of the functional polar lipids and cationic lipids to the mass ratio of phospholipids can be any value within the above range without special limitation, and can specifically be 0:100 (i.e., the polar lipids are only phospholipids), 1:99, 1.5:98.5, 2:98, 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, 98:2, 98.5:1.5, 99:1 or 100:0 (i.e., polar lipids are one or both of functional polar lipids and cationic lipids without phospholipids), which can be selected according to needs; the phospholipids are natural phospholipids and / or synthetic phospholipids well known to those skilled in the art, and there are no special restrictions, and can also be called main polar lipids. In the present invention, preferably, the phospholipids have a fatty acid chain length of more than 12 carbons, more preferably including but not limited to phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and One or more of sphingomyelin; the functional polar lipid is a lipid that helps stabilize the polar lipid monolayer membrane or provides a specific function, generally a natural sterol, a modified sterol or a modified phospholipid, and in the present invention preferably includes but is not limited to one or more of polyethylene glycol-modified sterols, biotin-modified sterols, amino acid-modified sterols, polypeptide-modified sterols, polysaccharide-modified sterols, nucleic acid-modified sterols, polyethylene glycol-modified phospholipids, biotin-modified phospholipids, amino acid-modified phospholipids, polypeptide-modified phospholipids, polysaccharide-modified phospholipids and nucleic acid-modified phospholipids; The cationic lipids are synthetic polar lipids with cations well known to those skilled in the art, and are not particularly limited. In the present invention, they preferably include but are not limited to (2,3-dioleoyl-propyl)-trimethylammonium-chloride, (2,3-dioleoyl-propyl)-trimethylamine, 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylamine hydrochloride, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinate One or more of: [N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride; in some embodiments provided by the present invention, the polar lipid is specifically one or more of distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, 1,2-dioleoylphosphatidylcholine, egg yolk lecithin, 1-hexadecanoyl-SN-glycerol-phosphocholine, phosphatidylethanolamine-polyethylene glycol 2000-amine and (2,3-dioleoyl-propyl)-trimethylammonium-chloride.

[0079] According to the present invention, the neutral lipids for preparing fat bodies are preferably hydrophobic neutral small molecules, more preferably including but not limited to one or more of triglycerides, wax esters, sterol esters, sterol esters, retinol esters, ether esters, polyhydroxyalkanoates and fat-soluble vitamins A, D, E, and K; the triglycerides are preferably triglycerides with a fatty acid chain length of more than 8 carbon atoms; the wax esters, sterol esters, sterol esters or retinol esters are preferably wax esters, sterol esters, sterol esters or retinol esters formed by fatty acids with a carbon chain length of more than 8 carbon atoms and short-chain, medium-chain or long-chain fatty alcohols; the fat-soluble vitamins are preferably one or more of tocopherol, phylloquinone and menaquinone; in some embodiments provided by the present invention, the neutral lipids are specifically one or more of triolein, fish oil, cholesterol and cholesterol oleate.

[0080] Polar lipids and neutral lipids are subjected to intermittent ultrasonic emulsification in a buffer solution to obtain a crude emulsion; the mass of the polar lipids is preferably 2.5% to 25% of the mass of the neutral lipids, more preferably 2.5% to 23%, and even more preferably 2.5% to 22%; in the present invention, the mass of the polar lipids is the mass of the neutral lipids and the concentration thereof can be any point value within the above range without special limitation, specifically 2.5%, 2.7%, 3.0%, 3.2%, 3.5%, 3.7%, 3.9%, 4.0%, 4.2%, 4.5%, 4.7%, 5.0%, 5.2%, 5.4%, 5.5%, 5.7%, 5.9%, 6.0%, 6.2%, 6.5%, 6.7%, 7.0%, 7.2%, 7.5%, 7.7%, 7.9%, 8.0%, 8.2%, 8.5%, 8.7%, 8.0%, 8.2%, 8.5%, 8.7%, 8.9%, 9.0%, 9.2%, 9.5%, 9.7%, 10.0%, 10.2%, 10.5%, 10.7%, 10.9%, 11.0%, 11.2%, 11.5%, 11.7%, 12.0%, 12.2%, 12.5%, 12.7%, 12.9%, 13.0%, 13.5%, 14.0%, 14.5%, 15%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 19.5%, 20.0%, 20.5%, 21.0%, 21.5%, 21.8%, 22.0%, 22. 5%, 23.0%, 23.5%, 24.0%, 24.5% or 25.0%; the buffer solution is any buffer solution known to those skilled in the art and is not particularly limited. In the present invention, HEPES buffer solution, PBS buffer solution or physiological saline solution are preferred; in some specific embodiments provided by the present invention, the HEPES buffer solution preferably includes 15-25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 80-120 mM KCl, 1.5-2.5mM MgCl2 and solvent water, more preferably comprising 20mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100mM KCl, 2mM MgCl2 and solvent water; the pH value of the HEPES buffer is preferably 7.2-7.6, more preferably 7.4; in other specific embodiments provided by the present invention, the PBS buffer preferably comprises 2-3mM KCl, 1.5-2.5mM KH2PO4, 120-150mM NaCl, 8-12mM Na2HPO4 and solvent water, more preferably comprising 2.5-2.8mM KCl, 1.8-2.2mM KH2PO4, 130-140mM NaCl, 9-11mM Na2HPO4 and solvent water, and more preferably comprising 2.68mM KCl, 2mM KH2PO4, 137mM NaCl, 10mM Na2HPO4 and solvent water; the pH value of the PBS buffer is preferably 7.2-7.6, more preferably 7.4; the physiological saline is specifically 0.9% NaCl (m / m).

[0081] In the present invention, since the ultrasonic probe can easily cause the fat body itself to have an irregular shape and easily form phospholipid membrane fragments, water bath ultrasound is preferably used; the present invention uses intermittent ultrasonic emulsification with a pause time during the ultrasonic process, which can avoid the risk of overheating of the emulsion and destruction of lipid components caused by continuous ultrasound; the amplitude of the intermittent ultrasonic emulsification treatment is preferably 40% to 100%, more preferably 50% to 90%, more preferably 50% to 80%, and most preferably 60% to 70%; in some embodiments provided by the present invention, an amplitude of 60% is specifically used for illustration; the present invention does not make special requirements on the power of the ultrasonic emulsification equipment, and in some embodiments provided by the present invention, a power of 250W is specifically used for illustration; the frequency of the intermittent ultrasonic emulsification treatment is preferably 20 to 50kHz, more preferably 20 to 45kHz, more preferably 30 to 45kHz, more preferably 35 to 45kHz, and most preferably 40kHz; in some embodiments provided by the present invention, the frequency of the intermittent ultrasonic emulsification treatment is specifically 40kHz; the intermittent ultrasonic The temperature of the emulsification treatment is preferably 25°C to 35°C; in the present invention, the temperature of the intermittent ultrasonic emulsification treatment can be any point value within the above range, specifically 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C; the time of the intermittent ultrasonic emulsification treatment is preferably 5 to 20 minutes, more preferably 8 to 20 minutes, more preferably 10 to 15 minutes, and most preferably 12 to 15 minutes; in the present invention, the intermittent ultrasonic emulsification treatment During the treatment process, the time of each ultrasonic treatment is preferably no more than 5 minutes; more specifically, the intermittent ultrasonic emulsification treatment is preferably: ultrasonic emulsification treatment for 1 to 5 minutes, then stopped for 10 to 60 seconds, and the time for the intermittent ultrasonic emulsification treatment is 5 to 20 minutes; in the present invention, the time of each ultrasonic emulsification treatment is preferably 1 to 3 minutes, more preferably 1 to 2 minutes; the time of each intermittent ultrasonic emulsification treatment is preferably 10 to 40 seconds, more preferably 10 to 30 seconds, and even more preferably 10 to 20 seconds.

[0082] In the present invention, the polar lipids and neutral lipids can be added in different ways.

[0083] In a specific embodiment provided by the present invention, polar lipids and neutral lipids are directly mixed, and then a buffer solution is added and subjected to intermittent ultrasonic emulsification treatment to obtain a coarse emulsion.

[0084] In a specific embodiment provided by the present invention, an organic solution containing polar lipids and an organic solution containing neutral lipids are mixed, the solvent is removed, and a buffer solution is added and subjected to intermittent ultrasonic emulsification to obtain a crude emulsion; the organic solvent in the above-mentioned organic solution can be an organic solvent well known to those skilled in the art, without any special restrictions, and specifically can be chloroform and / or ethanol in the present invention; the method for removing the solvent can be a method well known to those skilled in the art, without any special restrictions, and in the present invention, it is preferably to evaporate the organic solvent through a non-reactive gas flow, but if the organic solvent is ethanol, ethanol with a volume fraction of less than 8% (including 8%) can be retained; the non-reactive gas can be a non-reactive gas well known to those skilled in the art, without any special restrictions, and is preferably nitrogen in the present invention.

[0085] In a specific embodiment provided by the present invention, a neutral lipid is added to a buffer containing a polar lipid and subjected to intermittent ultrasonic emulsification to obtain a coarse emulsion; the ratio of polar lipid to buffer in the buffer containing polar lipid is preferably (100-1000) μg: (100-200) μL; in some embodiments provided by the present invention, the ratio of polar lipid to buffer in the buffer containing polar lipid is specifically 125 μg: (100-200) μL, 250 μg: (100-200) μL, 375 μg: (100-200) μL, 500 μg: (100-200) μL or 1000 μg: (100-200) μL; the buffer solution containing polar lipids is preferably prepared according to the following method: an organic solution of polar lipids is added to a reaction vessel, the organic solvent is evaporated cleanly by a non-reactive gas flow, and then a buffer solution is added. If the organic solvent is ethanol, 8% volume fraction or less (including 8%) of ethanol can be retained; the non-reactive gas can be a non-reactive gas well known to those skilled in the art and is not particularly limited. In the present invention, nitrogen is preferably used; the solvent in the organic solution containing polar lipids is preferably chloroform and / or ethanol.

[0086] In a specific embodiment provided by the present invention, the neutral lipid can be added in a dropwise manner, that is, part of the neutral lipid is added to the buffer containing polar lipid and then subjected to intermittent ultrasonic emulsification treatment, and the remaining neutral lipid is added in batches after one or more intervals in the intermittent ultrasonic emulsification process to obtain a crude emulsion; the preparation method of the buffer containing polar lipid is the same as described above, No further details will be given here; in the present invention, the amount of each batch of some neutral lipids and the remaining neutral lipids added in batches can be the same or different, and there is no special limitation. Preferably, the volume ratio of each batch of some neutral lipids to the remaining neutral lipids added in batches is 1: (0.5-2), more preferably 1: (0.5-1.5), and even more preferably 1: 1; the number of batches of the remaining neutral lipids and the number of intervals during the intermittent ultrasonic emulsification treatment can be the same or different, and there is no special limitation; in the present invention, it is preferred that the number of batches of the remaining neutral lipids is less than the number of intervals during the intermittent ultrasonic emulsification treatment, and it is more preferred that the intermittent ultrasonic emulsification treatment is continued for 7 to 10 cycles of ultrasonic intervals after the addition of the remaining neutral lipids in batches is completed.

[0087] In a specific embodiment provided by the present invention, an alcohol solution containing polar lipids is added to a buffer solution containing neutral lipids and subjected to intermittent ultrasonic emulsification treatment to obtain a coarse emulsion.

[0088] In another specific embodiment provided by the present invention, part of the alcohol solution containing polar lipids is added to a buffer solution containing neutral lipids and then subjected to intermittent ultrasonic emulsification treatment, and the remaining alcohol solution containing polar lipids is added in batches after the intermittent ultrasonic emulsification treatment to obtain a crude emulsion. In the present invention, the amount of each batch of the partial alcohol solution containing polar lipids and the remaining alcohol solution containing polar lipids added in batches can be the same or different, and there is no special limitation. Preferably, the volume ratio of the partial alcohol solution containing polar lipids to the remaining alcohol solution containing polar lipids added in batches is 1:(0.5-2), more preferably 1:(0.5-1.5), and even more preferably 1:1; the number of batches of the remaining alcohol solution containing polar lipids and the number of intervals during the intermittent ultrasonic emulsification treatment can be the same or different, and there is no special limitation; in the present invention, preferably, the number of batches of the remaining alcohol solution containing polar lipids is less than the number of intervals during the intermittent ultrasonic emulsification treatment, and more preferably, after the batch addition of the remaining alcohol solution containing polar lipids is completed, the intermittent ultrasonic emulsification treatment is continued for 7 to 10 cycles of ultrasonic intervals.

[0089] The crude emulsion is purified to obtain fat bodies; the resulting crude emulsion contains membrane structures resulting from phospholipid self-association, which should be removed during the separation and purification step. In the present invention, the principle that fat bodies contain a neutral lipid core, which generally results in a lower density than the buffer, can be exploited. The crude emulsion can be purified by stepwise centrifugation to remove membrane contamination in the crude emulsion and collect the fat bodies. The change in fat body density as a function of diameter is calculated as shown in formula (a).

[0090] Among them D Adiposome is the density of fat body, r Adiposome is the radius of the fat body, D Polar lipid and D Neutral lipid are the density of polar lipids and the density of neutral lipids, respectively. Polar lipid is the thickness of a fully hydrated polar lipid monolayer.

[0091] The calculation of the pseudo-gravitational acceleration (×g) required for the centrifugation process as a function of the fat body diameter is shown in formula (bd).

[0092] During the centrifugal process, fat bodies are mainly affected by three forces, namely buoyancy, viscous resistance and centrifugal force. When the three forces are balanced, the fat body radius r, fat body density ρ a , continuous phase (buffer) density ρ b , the centrifugal angular velocity ω, the effective centrifugal radius of the fat body x (the distance from the center of the fat body to the center of the centrifuge rotor), and the viscosity coefficient η of the continuous phase are as shown in formula b.

[0093] The floating coefficient s can be defined for the fat body according to the formula, and the corresponding analytical expression is formula c. Δρ is the difference between the continuous phase density and the fat body density.

[0094] Define the pseudo-gravitational acceleration in the centrifugal field as g'=ω 2 R, R is the effective radius of the centrifuge rotor, and the diameter of the fat body is d. The relationship between the distance traveled by the fat body in a fixed time during centrifugation and the pseudo-gravitational acceleration can be calculated using formula d.

[0095] Therefore, the required centrifugal force can be determined by determining the movement distance of the fat body and the centrifugal time in different centrifugal fields.

[0096] In a specific embodiment provided by the present invention, the crude emulsion is purified by step-by-step centrifugation to obtain fat bodies; the purification method cannot separate fat bodies of different particle sizes, and only fat bodies within a specific particle size range are retained; the step-by-step centrifugation is preferably controlled at a temperature within the range of 2°C to 25°C, more preferably within the range of 2°C to 20°C, more preferably within the range of 2°C to 15°C, more preferably within the range of 2°C to 10°C, more preferably within the range of 2°C to 6°C, and most preferably within the range of 4°C to 8°C. ℃; the stepwise centrifugation is specifically as follows: centrifuging the crude emulsion at 500-2000×g for 5-10 minutes to collect the lower emulsion; centrifuging the lower emulsion at 18000-21000×g for 5-10 minutes to obtain fat bodies; further specifically, the centrifugation rate of the crude emulsion is preferably 800-1500×g, more preferably 800-1200×g, and even more preferably 1000×g; further specifically, the centrifugation rate of the lower emulsion is preferably 19000-21000×g, more preferably 20000×g.

[0097] In another specific embodiment provided by the present invention, the crude emulsion is mixed with a density gradient centrifuge, and then a buffer is added and centrifuged stepwise to obtain a fat body; the stepwise centrifugation is preferably controlled at a temperature within the range of 2°C to 25°C, more preferably within the range of 2°C to 20°C, more preferably within the range of 2°C to 15°C, more preferably within the range of 2°C to 10°C, more preferably within the range of 2°C to 6°C, and most preferably at 4°C; the density gradient centrifuge comprises a density gradient medium and a buffer; the mass concentration of the density gradient medium in the density gradient centrifuge is preferably 50% to 80%, more preferably 50% to 60%; the density gradient medium is a substance that can provide a density gradient well known to those skilled in the art, and is not particularly limited. In the present invention, it preferably includes but is not limited to glycerol, sodium chloride, potassium chloride. , cesium chloride, sodium bromide, potassium bromide, iodixanol and sucrose; the volume of the density gradient centrifugation fluid is preferably 5% to 20% of the volume of the crude emulsion, more preferably 10% to 20%; the overall density of the emulsion can be increased by adding the density gradient centrifugation fluid; the type of the buffer solution is the same as described above and will not be repeated here; the amount of the buffer solution added is preferably such that the height of the emulsion phase and the buffer solution are close to or equal, and then stepwise centrifugation is performed; the stepwise centrifugation is preferably specifically as follows: first centrifugation at 1000 to 1600 × g for 5 to 10 minutes, then centrifugation at 16000 to 18000 × g for 5 to 10 minutes, and then centrifugation at 20000 to 21000 × g for 5 to 10 minutes, and fat bodies are collected from the top of the buffer solution after each centrifugation; in the present invention, further specifically, the buffer solution can be refilled as needed after each centrifugation to collect fat bodies during the stepwise centrifugation process. The use of density gradient medium can increase the overall density of the crude emulsion so that the fat bodies can float during centrifugation and be separated from membrane pollution. Different centrifugal speeds can be used to separate fat bodies with different particle size distributions, thereby obtaining fat bodies of different particle sizes.

[0098] In a specific embodiment provided by the present invention, the preparation can be carried out according to the schematic diagram of the preparation process of the fat body shown in FIG1 .

[0099] In another specific embodiment provided by the present invention, purification can also be performed by ultrafiltration centrifugation; specifically, the ultrafiltration centrifugation can use an exosome purification column to separate fat bodies with different average particle sizes; in some specific embodiments provided by the present invention, the step of purification by ultrafiltration centrifugation is specifically: the crude emulsion is subjected to a first centrifugation in an ultrafiltration centrifuge tube, after collecting the emulsion centrifuged by ultrafiltration, the remaining emulsion is subjected to a second centrifugation to obtain fat bodies; the molecular weight cutoff of the ultrafiltration centrifuge tube is preferably 50 to 200 kDa, more preferably 80 to 150 kDa, and even more preferably 100 kDa; the centrifugal force of the first centrifugation and the second centrifugation are each independently preferably 500-1000×g, more preferably 800-1000×g; the time of the first centrifugation and the second centrifugation are each independently preferably 5-10 min; the first centrifugation and the second centrifugation are preferably performed under low temperature conditions, specifically at 4°C; the collected emulsion centrifuged by ultrafiltration is preferably subjected to high-speed centrifugation to obtain fat bodies; the speed of the high-speed centrifugation is preferably 16000-20000×g; the time of the high-speed centrifugation is preferably 5-10 min; the high-speed centrifugation is preferably performed under low temperature conditions, specifically at 4°C.

[0100] The present invention also provides the use of the fat body prepared by the above method in preparing medicines or vaccines for preventing and treating diseases.

[0101] Specifically, the disease is cancer, and the cancer is: breast cancer, lung cancer, kidney cancer, laryngeal cancer, liver cancer, muscle tissue cancer, blood cancer, bone cancer, brain cancer, neck cancer, oral or nasal mucosal cancer, bladder cancer, central nervous system cancer, cervical cancer, colon cancer, endometrial cancer, external genital cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, genitourinary tract cancer, head cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, testicular cancer and / or thyroid cancer.

[0102] The present invention also provides a medicine or vaccine for preventing and treating diseases, which comprises pharmaceutically acceptable excipients and the fat body prepared by the above method.

[0103] In order to further illustrate the present invention, a method for preparing fat bodies provided by the present invention is described in detail below with reference to examples.

[0104] The reagents used in the following examples are all commercially available; the HEPES buffer in the examples includes 20.0 mM HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4; the PBS buffer used in the examples includes 2.68 mM KCl, 2 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4 and solvent water, pH 7.4.

[0105] Optimization of ultrasonic time for fat body crude emulsion

[0106] A 25 mg / ml 1,2-dioleoylphosphatidylcholine (DOPC) chloroform solution was prepared. A specific amount of DOPC (1,000 μg) was transferred to a 1.5 mL Eppendorf tube, and the chloroform was removed using a gentle stream of nitrogen. The dried DOPC was mixed with 100 μL of HEPES buffer to create a phospholipid buffer mixture. 5 μL of triolein (TOG) was added to the phospholipid buffer mixture. Thirteen samples were prepared per group, with triplicate runs. Ultrasonic emulsification was performed at 250 W, 40 kHz, and 60% amplitude at 30°C for 1 minute, with 30-second intervals between sonications, for a predetermined total sonication time. The specific sonication times for each sample were 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, and 20 minutes. Samples were taken at each time point to measure the average particle size and polydispersity index of the emulsion.

[0107] Figure 2 shows the particle size and polydispersity index of the emulsions obtained at different sonication times. (A) The particle size distribution of the emulsions obtained at different sonication times; (B) The polydispersity index of the emulsions obtained at different sonication times. n = 3, mean = ± SEM.

[0108] Example 1

[0109] A 25 mg / ml 1,2-dioleoylphosphatidylcholine (DOPC) chloroform solution was prepared, and specific amounts of DOPC (125 μg, 250 μg, 375 μg, 500 μg, and 1,000 μg) were transferred to a 1.5 mL Eppendorf tube and the chloroform was removed with a gentle stream of nitrogen. The dried DOPC was mixed with 100 μL of HEPES buffer. Then, 1 μL of triolein (TOG) was added to the buffer and the mixture was sonicated at 250 W, 40 kHz, 60% amplitude at 30 ± 5 ° C for 1 minute. The sonication was paused for 10 seconds while another 1 μL of TOG was added to the mixture. Then, the sonication was restarted for 1 minute. This process was repeated until 5 μL of TOG was finally added to the mixture. Seven further cycles of sonication were performed until a total sonication time of 12 minutes was obtained to prepare a crude fat body emulsion. Measure the absorbance of the crude emulsion (indicating the concentration of the emulsion) and select samples with high absorbance for separation and purification.

[0110] The crude emulsion was purified using fractional centrifugation to collect the fat bodies. First, 100 μL of the emulsion was mixed with 10 μL of HEPES buffer containing 50% glycerol to increase the density of the emulsion. Then, HEPES buffer was gently added to the top of the emulsion until the emulsion and buffer phases were close in height (the total volume of the emulsion and buffer was approximately 750 μL).

[0111] The suspension was subjected to gradient centrifugation, starting with 1,000-1,600 x g at 4°C for 5 min and fat body sample 1 was collected from the top of the buffer. If necessary, the buffer was gently refilled.

[0112] Centrifuge again at 16,000-18,000 × g at 4°C for 5 minutes and collect fat body sample 2 from the top of the buffer. Gently refill the buffer if necessary.

[0113] Finally, the cells were centrifuged at 20,000-21,000 × g for 5 min at 4°C and fat body samples were collected from the top of the buffer solution.

[0114] The collected fat body samples 1, 2, and 3 were observed for their microscopic morphology using an optical microscope and an electron microscope, their average particle size and polydispersity were measured using a dynamic light scattering particle size analyzer, and their lipid composition and purity were detected using thin layer chromatography.

[0115] Figure 3 shows crude emulsions prepared at different lipid ratios. 375 μg and 500 μg crude emulsions were used to isolate and purify fat bodies, and their particle size distribution was measured. A control group (2,000 μg DOPC) containing the same phospholipid dosage reported in the literature (ACS Nano 2016, 10, 3, 3312–3322) was used. A represents the OD600 value of the purified fat bodies produced using different amounts of DOPC. (The purification conditions here were centrifugation at 1,000 × g, 4°C for 5 minutes, collection of the lower emulsion layer, and discarding of the upper, large lipid layer. The collected emulsion was then centrifuged at 20,000 × g, 4°C for 5 minutes, and the precipitated lipid particles in the lower layer were discarded. The upper emulsion layer was the purified fat body.) B represents the correlation between the required fat body density and fat body diameter in the centrifugal selection method for the purification of fat bodies of different sizes. The fitting method is shown in formula (a). C is the fitted correlation curve between pseudo-gravitational acceleration (×g) and fat body diameter. The calculation method for pseudo-gravitational acceleration, centrifugation time for collecting fat bodies with determined diameters, and fat body travel distance is based on the formula bd. D is the diameter of fat bodies collected at different centrifugal forces, where 375 μg of DOPC was used to produce fat bodies. d0 is an unpurified emulsion sample. d1, d2, and d3 represent different centrifugation processes. d1 is a sample collected at 1,000 × g, d2 is a sample collected at 1,000 × g followed by a centrifugation at 16,000 × g, and d3 is a sample collected at 1,000 × g and 16,000 × g, respectively, followed by a centrifugation at 20,000 × g. d4, d5, and d6 represent different centrifugal forces and processes. d4 is a sample collected at 1,300 × g, d5 is sample collection at 1,300 × g followed by 16,000 × g, d6 is sample collection at 1,300 × g and 16,000 × g respectively followed by centrifugation at 20,000 × g, d7, d8, and d9 represent different centrifugal forces and processes, d7 is sample collection at 1,600 × g, d8 is sample collection at 1,600 × g followed by centrifugation at 16,000 × g, and d9 is sample collection at 1,600 × g and 16,000 × g respectively followed by centrifugation at 20,000 × g.E is the diameter of fat bodies collected at different centrifugal forces using 500 μg DOPC to produce fat bodies, where e0 is an unpurified emulsion sample, e1, e2, and e3 represent different centrifugation processes, e1 is a sample collected by centrifugation at 1,000 × g, e2 is a sample collected by centrifugation at 1,000 × g followed by centrifugation at 16,000 × g, e3 is a sample collected by centrifugation at 1,000 × g and 16,000 × g respectively followed by centrifugation at 20,000 × g, e4, e5, and e6 represent different centrifugal forces and processes, e4 is a sample collected by centrifugation at 1,300 × g Samples were collected by centrifugation. e5: 1,300 × g followed by 16,000 × g; e6: 1,300 × g and 16,000 × g, followed by 20,000 × g; e7, e8, and e9 represent different centrifugal forces and procedures. e7: 1,600 × g; e8: 1,600 × g followed by 16,000 × g; and e9: 1,600 × g and 16,000 × g, followed by 20,000 × g. n = 3, mean = ± SEM.

[0116] Figure 4 provides the microscopic morphology and average particle size of fat bodies collected under different centrifugation conditions in the 500 μg group. (A) Fat bodies collected at 1,300 × g. (B) Fat bodies collected at 16,000 × g. (C) Fat bodies collected at 20,000 × g. Fat bodies were stained with LipidTOX Red (1:1,000, v / v). (a) LipidTox Red channel, (b) DIC channel, (c) merged channel. (d) Average diameter determined from three independent samples (n = 3), mean ± SEM. Scale bar, 2 μm.

[0117] Figure 5 shows the transmission electron microscopic morphology of fat bodies collected under different centrifugation conditions in the 500 μg group. Liposomes were prepared by transferring 500 μg of DOPC dissolved in chloroform to a 1.5 mL Eppendorf tube and drying it with a gentle stream of nitrogen. The dried phospholipids were mixed with 200 μL of HEPES buffer and 5 μL of triolein. The mixture was sonicated at 250 W, 40 kHz, and 60% amplitude at 30 ± 5°C for 1 minute, followed by 1 minute off, for 12 cycles. The prepared fat body suspension was collected for further use. Liposome samples were prepared by transferring 80 μL of a 25 mg / mL DOPC chloroform solution (containing 2,000 μg of DOPC) to a new 1.5 mL Eppendorf tube and evaporating the chloroform with a gentle stream of nitrogen. The dried DOPC was mixed with 100 μL of HEPES buffer to prepare a phospholipid buffer mixture. The phospholipid buffer mixture was then sonicated. The mixture was sonicated at 250W, 40kHz, and 60% amplitude at 30±5°C for 1 minute, followed by a 10-second pause. This process was repeated until a total sonication time of 12 minutes was achieved to prepare a liposome suspension. Images of liposomes or liposome samples were taken. Images A and B show liposomes collected by centrifugation at 1,300×g. The scale bar in A is 10μm, and the scale bar in B is 500nm. Images C and D show liposomes collected by centrifugation at 1,300×g followed by 16,000×g. The scale bar in C is 10μm, and the scale bar in D is 500nm. Images E and F show liposomes collected by centrifugation at 1,300×g, 16,000×g, and then 20,000×g. The scale bar in E is 10μm, and the scale bar in F is 500nm. Images G and H show liposome controls. The scale bar in G is 1μm, and the scale bar in H is 200nm.

[0118] Figure 6 provides a graph showing the calculation results of lipid utilization of fat bodies, wherein A is a graph showing TOG quantification by thin-layer chromatography, a2 to c2 are fat bodies collected by centrifugation at 1,000 × g, 1,300 × g, and 1,600 × g, respectively, and 375 μg of DOPC was used to produce fat bodies; d2 to f2 are fat bodies collected by centrifugation at 1,000 × g, 1,300 × g, and 1,600 × g, respectively, and 500 μg of DOPC was used to produce fat bodies. Total TOG was prepared using 4.6 mg (5 μL) of TOG, not mixed with DOPC, but added directly to the same volume of buffer, followed by the same sonication and lipid extraction procedures. B is a quantification of DOPC by thin-layer chromatography. Figures a1-c1 show liposomes collected by centrifugation at 1,000 × g, 1,300 × g, and 1,600 × g, respectively, and prepared with 375 μg of DOPC. Figures d1-f1 show liposomes collected by centrifugation at 1,000 × g, 1,300 × g, and 1,600 × g, respectively, and prepared with 500 μg of DOPC. Total DOPC was prepared using 500 μg of DOPC, not mixed with TOG, but added directly to the same volume of buffer, followed by the same sonication and lipid extraction procedures. C shows the yields of DOPC and TOG calculated using ImageJ based on grayscale intensity on the TLC plate. D is the ratio of DOPC to total lipids. For all quantitative measurements, n = 3. Under these conditions, the neutral lipid utilization rate of liposome fractions collected after centrifugation at 1,000 × g, 1,300 × g, or 1,600 × g and then at 16,000 × g was >40%, and the phospholipid utilization rate was >30%. This phospholipid utilization rate was nearly fourfold higher than that of published methods. The ratio of phospholipids to neutral lipids also showed that the actual liposome purity was consistent with the predicted value. Thin-layer chromatography was performed using a neutral lipid eluent (n-hexane: ether: acetic acid = 80:20:1, v / v / v) and, after drying, then developing with a phospholipid eluent (chloroform: methanol: acetic acid: water = 75:13:9:3, v / v / v / v) to a depth of 1 / 5 to 1 / 4 the neutral lipid eluent. After sufficient drying, the samples were stained with saturated iodine vapor for 30 minutes to visualize the lipid composition.

[0119] Figure 7 shows the results of a fat body stability test. Fat bodies were produced using 500 μg of DOPC. Fat bodies collected by centrifugation at 1,300 × g and then 16,000 × g were sealed and stored at 4°C. The average diameter of the fat bodies was measured using DLS at predetermined intervals (n = 9). Figure 7 shows that the stability of the fat bodies collected by centrifugation at 16,000 × g did not change significantly over 90 days.

[0120] Example 2

[0121] A phospholipid mixture (DOPC (98.5%) + DSPE-PEG (2000) Amine (1.5%), mol / mol) was first dissolved in ethanol. Ultrasonication and heating were used to adjust the ethanolic phospholipid solution concentrations to 64, 95, and 127 mM, respectively. 5 μl of triolein (TOG) was added to 100 μl of HEPES buffer. The ethanolic solution of the dissolved phospholipid mixture was then added dropwise to the TOG buffer mixture, 1 μl at a time. The mixture was sonicated at 250 W, 40 kHz, and 60% amplitude at 30 ± 5°C for 1 minute. The sonication was paused for 10 seconds. The ethanol solution was then added dropwise, sonicating for 1 minute and pausing until all 4 μl of the ethanolic phospholipid solution had been added. An additional 2 μl of pure ethanol was then added, resulting in phospholipid additions of 0.25 μmol, 0.38 μmol, and 0.51 μmol, respectively, corresponding to phospholipid / neutral lipid ratios of 5%, 7.5%, and 10% (mol%). Repeat until the total ultrasonic treatment time is 15 minutes to prepare a fat body crude emulsion.

[0122] The crude emulsion was purified by ultrafiltration centrifugation to collect the fat bodies. An ExoEasy spin column (QIAGEN, Germany) with a molecular weight cutoff of 100 kDa was used. 800 μl of crude fat body emulsion was transferred to the column and centrifuged at 1,000 × g for 5 minutes at 4°C. The lower emulsion (approximately 50% to 60% of the total volume) was collected into a new 1.5 ml EP tube. The upper emulsion was further centrifuged (1,000 × g for 5 minutes at 4°C). The remaining emulsion was designated as Fraction 1. The lower emulsion was then centrifuged again at 16,000 × g for 5 minutes at 4°C to separate the upper fat body layer from the lower dilute emulsion (approximately 80% of the total volume). The upper fat body layer was designated as Fraction 2, and the lower dilute emulsion was designated as Fraction 3.

[0123] The collected fat body sample components 1, 2, and 3 were observed for their microscopic morphology using an optical microscope, and their average particle size and polydispersity were measured using a dynamic light scattering particle size analyzer.

[0124] FIG8 is a schematic diagram of the process for preparing fat bodies according to Example 2 of the present invention.

[0125] Figure 9 shows that Fractions 1 and 2 are rich in fat bodies and appear as white emulsions, while Fraction 3 contains very few fat bodies and is nearly transparent. OD600 (the absorbance of the emulsion at 600 nm) indicates that the concentration of Fraction 1 is much higher than that of Fraction 2 (under conditions where the emulsion droplet size is similar).

[0126] Figures 10-12 show the microscopic morphology of fat bodies prepared from three different phospholipid concentrations. Figure 10 shows the microscopic morphology of fat body fractions with a 5.0% phospholipid neutral lipid ratio: (A) Fraction 1, (B) Fraction 2, and (C) Fraction 3. From left to right, the images are bright field (DIC), Nile Red staining, and merged channels. Scale bar: 5 μm.

[0127] Figure 11 shows the microscopic morphology of different fat body fractions with a 7.5% phospholipid-neutral lipid ratio. (A) Fraction 1, (B) Fraction 2, and (C) Fraction 3. From left to right, bright field (DIC), Nile Red staining, and merged channels are shown. Scale bar: 5 μm.

[0128] Figure 12 shows the microscopic morphology of fat body fractions at different 10.0% phospholipid-neutral lipid ratios. (A) Fraction 1, (B) Fraction 2, and (C) Fraction 3. From left to right, the images are bright field (DIC), Nile Red staining, and merged channels. Scale bar: 5 μm.

[0129] Figures 10-12 show that Fractions 1 and 2 in each group are uniform spherical particles. However, under the same dilution conditions, Fraction 3, despite having a smaller droplet volume, exhibits Nile Red staining of the neutral lipid core within the spherical particles. Figure 13 provides the particle size distribution of different adipocytes. As the phospholipid dosage increases, the average particle size of Fraction 1 decreases, while that of Fractions 2 and 3 shows little change. This method demonstrates the applicability of ultrafiltration separation to the separation and purification of adipocytes.

[0130] Figure 13 shows the particle size distribution of fat body fractions at different phospholipid neutral lipid ratios. (A) Average particle size of fat body fractions at 5.0%, 7.5%, and 10.0% phospholipid neutral lipid ratios. (B) Monodispersity of fat body fractions at 5.0%, 7.5%, and 10.0% phospholipid neutral lipid ratios. F1 represents Fraction 1, F2 represents Fraction 2, and F3 represents Fraction 3.

[0131] Example 3

[0132] Prepare a 32mM 1,2-dioleoylphosphatidylcholine (DOPC) chloroform solution, transfer 40μl of the DOPC chloroform solution to a 1.5ml Eppendorf tube, and remove the chloroform with a gentle stream of nitrogen. Mix the dried DOPC with 100μl of PBS buffer. Then, add 5μl of neutral lipid (TOG or fish oil) to the buffer, and sonicate the mixture at 250W, 40kHz, 60% amplitude at 30±5°C for 1 minute. Pause the sonication for 10 seconds and then restart the sonication for 1 minute. Repeat this process until the total sonication time is 15 minutes to prepare a crude fat body emulsion.

[0133] The crude emulsion was collected directly and aliquoted into 1.5 ml Eppendorf tubes, 100 μl per tube. Centrifuge at 1,000 × g for 5 minutes to collect the lower emulsion (approximately 80% of the total volume). Centrifuge the collected emulsion again at 20,000 × g for 5 minutes to remove the lower precipitate (if any). The recollected emulsion is the fat body delivery platform product.

[0134] The micromorphology of fat body samples was observed by optical microscope and electron microscope, and the average particle size and polydispersity were measured by dynamic light scattering particle size analyzer.

[0135] Figure 14 provides the microscopic morphology of the TOG fat body prepared in Example 3. Both fluorescence micrographs and electron micrographs show that the TOG fat body has a neutral lipid core and is spherical overall. (A) Fluorescence micrograph of TOG fat body, from left to right: bright field (DIC), Nile Red staining (Nile Red), and merged channel (Merged), scale bar = 5 μm; (B) Transmission electron microscopy positive staining of TOG fat body, scale bar = 5 μm; (C) Transmission electron microscopy positive staining of fat body, scale bar = 0.5 μm; (D) The average particle size of TOG fat body is 145.1 ± 1.9 nm, and the polydispersity index is 0.130-0.182.

[0136] Figure 15 provides the microscopic morphology of the fish oil adipomes prepared in Example 3. Both fluorescence and electron micrographs show that the fish oil adipomes have a neutral lipid core and are spherical overall. This demonstrates that this method can successfully prepare adipomes. (A) Fluorescence micrograph of fish oil adipomes, from left to right: bright field (DIC), Nile Red staining, and merged channels (scale bar = 5 μm); (B) Transmission electron microscopy of fish oil adipomes, scale bar = 5 μm; (C) Transmission electron microscopy of fish oil adipomes, scale bar = 0.5 μm; (D) The average particle size of the adipomes is 130.0 ± 3.1 nm, and the polydispersity index is 0.072-0.127.

[0137] Example 4

[0138] Lipids of varying polarity were selected as surfactants: dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), egg yolk phosphatidylcholine (Egg PC), (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), and 1-hexadecanoyl-sn-glycerol-phosphocholine (Lyso PC). Each polar lipid was prepared as a 32 mM stock solution in chloroform. 40 μl of each chloroform stock solution was transferred to a 1.5 ml Eppendorf tube, and the chloroform was removed using a gentle stream of nitrogen. The dried lipids of varying polarity were mixed with 100 μl of PBS buffer. Then, 5 μl of a neutral lipid (TOG) was added to the buffer, and the mixture was sonicated at 40 kHz, 60% amplitude, and 30 ± 5°C for 1 minute. The sonication was paused for 10 seconds and then resumed for 1 minute. This process was repeated until a total sonication time of 15 minutes was achieved, resulting in a crude fat body emulsion.

[0139] The crude emulsion was collected directly and aliquoted into 1.5 ml Eppendorf tubes, 100 μl per tube. Centrifuge at 1,000 × g for 5 minutes to collect the lower emulsion (approximately 80% of the total volume). Centrifuge the collected emulsion again at 20,000 × g for 5 minutes to remove the lower precipitate (if any). The recollected emulsion is the fat body delivery platform product.

[0140] The average particle size and polydispersity were measured using a dynamic light scattering particle size analyzer, and the lipid composition of the fatsome delivery platform was identified using thin-layer chromatography.

[0141] Figure 16 shows the particle size distribution of liposome components synthesized from different polar lipids and neutral lipids. (A) Average particle size of liposomes synthesized from DPPC, DSPC, Egg PC, DOTAP, and Lyso PC with TOG. (B) Polydispersity coefficient of liposomes synthesized from DPPC, DSPC, Egg PC, DOTAP, and Lyso PC with TOG. (C) Lipid composition of liposomes synthesized from DPPC, DSPC, Egg PC, DOTAP, and Lyso PC with TOG. Thin-layer chromatography (TLC) was performed using a neutral lipid eluent (n-hexane: ether: acetic acid = 80:20:1, v / v / v). After drying, the eluent was then developed with a phospholipid eluent (chloroform: methanol: acetic acid: water = 75:13:9:3, v / v / v / v) to a depth 1 / 5 to 1 / 4 of the neutral lipid eluent. After sufficient drying, the eluent was stained with saturated iodine vapor for 30 minutes to visualize the lipid composition.

[0142] Example 5

[0143] Prepare a 32 mM 1,2-dioleoylphosphatidylcholine (DOPC) chloroform solution and a 32 mM cholesterol chloroform solution. Transfer 40 μl of DOPC, 32 μl of DOPC and 8 μl of cholesterol, 24 μl of DOPC and 16 μl of cholesterol, 16 μl of DOPC and 24 μl of cholesterol, and 8 μl of DOPC and 32 μl of cholesterol to a 1.5 ml Eppendorf tube, respectively. Remove the chloroform with a gentle stream of nitrogen. Mix the dried polar lipid with 100 μl of PBS buffer. Then, add 5 μl of neutral lipid (TOG) to the buffer, and sonicate the mixture at 40 kHz, 60% amplitude, and 30 ± 5°C for 1 minute. Pause the sonication for 10 seconds and then resume sonication for 1 minute. Repeat this process until the total sonication time is 15 minutes to prepare a crude fat body emulsion.

[0144] The crude emulsion was collected directly and aliquoted into 1.5 ml Eppendorf tubes, 100 μl per tube. Centrifuge at 1,000 × g for 5 minutes to collect the lower emulsion (approximately 80% of the total volume). Centrifuge the collected emulsion again at 20,000 × g for 5 minutes to remove the lower precipitate (if any). The collected emulsion is the fat body delivery platform product.

[0145] The average particle size and polydispersity were measured using a dynamic light scattering particle size analyzer, and the lipid composition of the fatsome delivery platform was identified using thin-layer chromatography.

[0146] Figure 17 shows the particle size distribution of liposome components synthesized with different DOPC and cholesterol ratios and neutral lipids. (A) Average particle size of liposomes synthesized with 0%, 20%, 40%, 60%, and 80% cholesterol and the corresponding DOPC and TOG ratios. (B) Polydispersity coefficient of liposomes synthesized with 0%, 20%, 40%, 60%, and 80% cholesterol and the corresponding DOPC and TOG ratios. (C) Lipid composition of liposomes synthesized with 0%, 20%, 40%, 60%, and 80% cholesterol and the corresponding DOPC and TOG ratios. Thin-layer chromatography was performed using a neutral lipid eluent (n-hexane: ether: acetic acid = 80:20:1, v / v / v), air-dried, and then developed with a phospholipid eluent (chloroform: methanol: acetic acid: water = 75:13:9:3, v / v / v / v) to a depth of 1 / 5 to 1 / 4 the neutral lipid eluent. After fully drying, stain with saturated iodine vapor for 30 minutes to observe the lipid components.

[0147] Example 6

[0148] Prepare a chloroform solution of 32mM dipalmitoylphosphatidylcholine (DPPC) and a chloroform solution of 32mM cholesterol. Transfer 40μl of DPPC, 32μl of DPPC and 8μl of cholesterol, 24μl of DPPC and 16μl of cholesterol, 16μl of DPPC and 24μl of cholesterol, and 8μl of DPPC and 32μl of cholesterol to a 1.5ml Eppendorf tube and remove the chloroform with a gentle stream of nitrogen. Mix the dried polar lipid with 100μl of PBS buffer. Then, add 5μl of neutral lipid (TOG) to the buffer and sonicate the mixture at 40kHz, 60% amplitude at 30±5°C for 1 minute. Pause the sonication for 10 seconds and then restart the sonication for 1 minute. Repeat this process until the total sonication time is 15 minutes to obtain a crude fat body emulsion.

[0149] The crude emulsion was collected directly and aliquoted into 1.5 ml Eppendorf tubes, 100 μl per tube. Centrifuge at 1,000 × g for 5 minutes to collect the lower emulsion (approximately 80% of the total volume). Centrifuge the collected emulsion again at 20,000 × g for 5 minutes to remove the lower precipitate (if any). The recollected emulsion is the fat body delivery platform product.

[0150] The average particle size and polydispersity were measured using a dynamic light scattering particle size analyzer, and the lipid composition of the fatsome delivery platform was identified using thin-layer chromatography.

[0151] Figure 18 shows the particle size distribution of liposome components synthesized with different DPPC and cholesterol ratios and neutral lipids. (A) Average particle size of liposomes synthesized with 0%, 20%, 40%, 60%, and 80% cholesterol and the corresponding ratios of DPPC and TOG. (B) Polydispersity coefficient of liposomes synthesized with 0%, 20%, 40%, 60%, and 80% cholesterol and the corresponding ratios of DPPC and TOG. (C) Lipid composition of liposomes synthesized with 0%, 20%, 40%, 60%, and 80% cholesterol and the corresponding ratios of DPPC and TOG. Thin-layer chromatography was performed using a neutral lipid eluent (n-hexane: ether: acetic acid = 80:20:1, v / v / v), air-dried, and then developed with a phospholipid eluent (chloroform: methanol: acetic acid: water = 75:13:9:3, v / v / v / v) to a depth of 1 / 5 to 1 / 4 of the neutral lipid eluent. After fully drying, stain with saturated iodine vapor for 30 minutes to observe the lipid components.

[0152] Example 7

[0153] Prepare a chloroform solution of 32mM distearoylphosphatidylcholine (DSPC) and a chloroform solution of 32mM cholesterol. Transfer 40μl of DSPC, 32μl of DSPC and 8μl of cholesterol, 24μl of DSPC and 16μl of cholesterol, 16μl of DSPC and 24μl of cholesterol, and 8μl of DSPC and 32μl of cholesterol to a 1.5ml Eppendorf tube and remove the chloroform with a gentle stream of nitrogen. Mix the dried polar lipid with 100μl of PBS buffer. Then, add 5μl of neutral lipid (TOG) to the buffer and sonicate the mixture at 40kHz, 60% amplitude at 30±5°C for 1 minute. Pause the sonication for 10 seconds and then restart the sonication for 1 minute. Repeat this process until the total sonication time is 15 minutes to obtain a crude fat body emulsion.

[0154] The crude emulsion was collected directly and aliquoted into 1.5 ml Eppendorf tubes, 100 μl per tube. Centrifuge at 1,000 × g for 5 minutes to collect the lower emulsion (approximately 80% of the total volume). Centrifuge the collected emulsion again at 20,000 × g for 5 minutes to remove the lower precipitate (if any). The recollected emulsion is the fat body delivery platform product.

[0155] The average particle size and polydispersity were measured using a dynamic light scattering particle size analyzer, and the lipid composition of the fatsome delivery platform was identified using thin-layer chromatography.

[0156] Figure 19 shows the particle size distribution of liposome components synthesized with different DSPC and cholesterol ratios and neutral lipids. (A) Average particle size of liposomes synthesized with 0%, 20%, 40%, 60%, and 80% cholesterol and corresponding DSPC and TOG ratios. (B) Polydispersity coefficient of liposomes synthesized with 0%, 20%, 40%, 60%, and 80% cholesterol and corresponding DSPC and TOG ratios. (C) Lipid composition of liposomes synthesized with 0%, 20%, 40%, 60%, and 80% cholesterol and corresponding DSPC and TOG ratios. Thin-layer chromatography was performed using a neutral lipid eluent (n-hexane: ether: acetic acid = 80:20:1, v / v / v), air-dried, and then developed with a phospholipid eluent (chloroform: methanol: acetic acid: water = 75:13:9:3, v / v / v / v) to a depth of 1 / 5 to 1 / 4 the neutral lipid eluent. After fully drying, stain with saturated iodine vapor for 30 minutes to observe the lipid components.

[0157] Example 8

[0158] Prepare 32 mM chloroform solutions of DOPC, DPPC, and DSPC, as well as a 32 mM chloroform solution of cholesteryl oleate (CE). Transfer 40 μl of each of DOPC, DPPC, and DSPC to a 1.5 ml Eppendorf tube. Transfer 240 μl of the cholesteryl oleate chloroform solution to each Eppendorf tube. Remove the chloroform with a gentle stream of nitrogen. Mix the dried lipid mixture with 100 μl of PBS buffer. Then, sonicate the mixture at 40 kHz, 60% amplitude, at 30 ± 5°C for 1 minute. Pause the sonication for 10 seconds and then resume sonication for 1 minute. Repeat this process until the total sonication time is 15 minutes to produce a crude fat body emulsion.

[0159] The crude emulsion was collected directly and aliquoted into 1.5 ml Eppendorf tubes, 100 μl per tube. Centrifuge at 1,000 × g for 5 minutes to collect the lower emulsion (approximately 80% of the total volume). Centrifuge the collected emulsion again at 20,000 × g for 5 minutes to remove the lower precipitate (if any). The recollected emulsion is the fat body delivery platform product.

[0160] The average particle size and polydispersity were measured using a dynamic light scattering particle size analyzer, and the lipid composition of the fatsome delivery platform was identified using thin-layer chromatography.

[0161] Figure 20 shows the particle size distribution of liposome components synthesized from different phospholipids and cholesterol oleate. (A) Average particle size of liposomes synthesized from DOPC, DPPC, and DSPC with cholesterol oleate, respectively. (B) Polydispersity coefficient of liposomes synthesized from DOPC, DPPC, and DSPC with cholesterol oleate, respectively. (C) Lipid composition of liposomes synthesized from DOPC, DPPC, and DSPC with cholesterol oleate, respectively. Thin-layer chromatography (TLC) was performed using a neutral lipid eluent (n-hexane: ether: acetic acid = 80:20:1, v / v / v). After drying, the samples were then developed with a phospholipid eluent (chloroform: methanol: acetic acid: water = 75:13:9:3, v / v / v / v) to a depth of 1 / 5 to 1 / 4 of the neutral lipid eluent. After sufficient drying, the samples were stained with saturated iodine vapor for 30 minutes to visualize the lipid composition.

[0162] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing fat bodies, characterized in that: The following steps are involved: S1) treating polar lipids and neutral lipids in a buffer solution by intermittent ultrasonic emulsification to obtain a crude emulsion; S2) purifying the crude emulsion to obtain fat bodies.

2. The preparation method according to claim 1, characterized in that The amplitude of the intermittent ultrasonic emulsification treatment is 40% to 100%; the frequency of the intermittent ultrasonic emulsification treatment is 20 to 50 kHz; the temperature of the intermittent ultrasonic emulsification treatment is 25°C to 35°C; the time of the intermittent ultrasonic emulsification treatment is 5 to 20 minutes, more preferably 8 to 20 minutes, more preferably 10 to 15 minutes, and most preferably 12 to 15 minutes.

3. The preparation method according to claim 1, characterized in that The intermittent phacoemulsification treatment is specifically as follows: The ultrasonic emulsification treatment is performed for 1 to 5 minutes, and then stopped for 20 to 60 seconds, and this cycle is repeated until the intermittent ultrasonic emulsification treatment time is 5 to 20 minutes.

4. The preparation method according to claim 1, characterized in that The step S1) is specifically as follows: Polar lipids and neutral lipids are directly mixed, and then a buffer solution is added and subjected to intermittent ultrasonic emulsification treatment to obtain a coarse emulsion.

5. The preparation method according to claim 1, characterized in that The step S1) is specifically as follows: After mixing an organic solution containing polar lipids with an organic solution containing neutral lipids, removing the solvent, adding a buffer solution and performing intermittent ultrasonic emulsification treatment, a coarse emulsion is obtained.

6. The preparation method according to claim 1, characterized in that The step S1) is specifically as follows: Neutral lipids are added to a buffer solution containing polar lipids and subjected to intermittent ultrasonic emulsification to obtain a coarse emulsion.

7. The preparation method according to claim 1, characterized in that The step S1) is specifically as follows: Part of the neutral lipids is added to a buffer solution containing polar lipids and then subjected to intermittent ultrasonic emulsification treatment, and the remaining neutral lipids are added in batches after one or more intervals in the intermittent ultrasonic emulsification process to obtain a coarse emulsion.

8. The preparation method according to claim 1, characterized in that The step S1) is specifically as follows: The alcohol solution containing polar lipids was added to the buffer solution containing neutral lipids and the mixture was separated by intermittent ultrasonic emulsification. The crude emulsion was obtained by chemical treatment.

9. The preparation method according to claim 1, characterized in that The step S1) is specifically as follows: A portion of the alcohol solution containing polar lipids is added to a buffer solution containing neutral lipids and then subjected to intermittent ultrasonic emulsification treatment, and the remaining alcohol solution containing polar lipids is added in batches after one or more intervals in the intermittent ultrasonic emulsification process to obtain a coarse emulsion.

10. The preparation method according to claim 1, characterized in that The step S2) is specifically as follows: Purifying the crude emulsion by stepwise centrifugation to obtain fat bodies; The stepwise centrifugation specifically comprises the following steps: centrifuging the crude emulsion at 500-2000×g for 5-10 minutes to collect the lower emulsion; and centrifuging the lower emulsion at 18000-21000×g for 5-10 minutes to obtain the fat body.

11. The preparation method according to claim 1, characterized in that The step S2) is specifically as follows: The crude emulsion is mixed with a density gradient centrifuge solution, and then a buffer solution is added and centrifuged stepwise to obtain fat bodies; the density gradient centrifuge solution comprises a density gradient medium and a buffer solution; the mass concentration of the density gradient medium in the density gradient centrifuge solution is 50% to 80%; The volume of the density gradient centrifugation fluid is 5% to 20% of the volume of the crude emulsion.

12. The preparation method according to claim 11, characterized in that The stepwise centrifugation is specifically as follows: First, centrifuge at 1000-1600×g for 5-10 min, then at 16000-18000×g for 5-10 min, and then at 20000-21000×g for 5-10 min. Collect fat bodies from the top of the buffer after each centrifugation.

13. The preparation method according to claim 1, characterized in that The step S2) specifically comprises: performing a first centrifugation on the crude emulsion in an ultrafiltration centrifuge tube, collecting the emulsion centrifuged by ultrafiltration, and performing a second centrifugation on the remaining emulsion to obtain fat bodies; The collected emulsion obtained by ultrafiltration centrifugation is subjected to high-speed centrifugation to obtain fat bodies.

14. The preparation method according to claim 13, characterized in that The centrifugal force of the first centrifugation and the second centrifugation are each independently 500 to 1000×g; The time of the first centrifugation and the second centrifugation is independently 5 to 10 minutes; The first centrifugation and the second centrifugation are both performed under low temperature conditions; The high-speed centrifugation speed is 16000-20000×g; The time of the high-speed centrifugation is 5 to 10 minutes.

15. The preparation method according to claim 1, characterized in that The polar lipids include one or more of phospholipids, functional polar lipids and cationic lipids; The phospholipid is selected from one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and sphingomyelin; The functional polar lipid is selected from one or more of polyethylene glycol-modified sterols, biotin-modified sterols, amino acid-modified sterols, polypeptide-modified sterols, polysaccharide-modified sterols, nucleic acid-modified sterols, polyethylene glycol-modified phospholipids, biotin-modified phospholipids, amino acid-modified phospholipids, polypeptide-modified phospholipids, polysaccharide-modified phospholipids and nucleic acid-modified phospholipids; The cationic lipid is selected from one or more of (2,3-dioleoyl-propyl)-trimethylammonium-chloride, (2,3-dioleoyl-propyl)-trimethylamine, 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylamine hydrochloride, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl](nickel salt) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride; The neutral lipid is selected from one or more of triglycerides, wax esters, sterol esters, sterol esters, retinol esters, ether esters, polyhydroxyalkanoates, vitamin A, vitamin D, vitamin E, and vitamin K.

16. The preparation method according to claim 13, characterized in that The polar lipids include one or both of functional polar lipids and cationic lipids and phospholipids; The mass ratio of the sum of the mass of one or both of the functional polar lipid and the cationic lipid to the mass of the phospholipid is (0-100):(100-0), more preferably (0-80):(100-20), further preferably (0-60):(100-40), and further preferably (0-50):(100-50).

17. The preparation method according to claim 1, characterized in that The mass of the polar lipids is 2.5% to 25% of the mass of the neutral lipids, more preferably 2.5% to 23%, and even more preferably 2.5% to 22%.

18. Fat bodies prepared according to the method according to any one of claims 1 to 17.

19. Use of the fat body according to claim 18 in preparing medicines or vaccines for preventing and treating diseases.

20. The use according to claim 19, characterized in that The disease is cancer.

21. A drug or vaccine, characterized in that The medicine or vaccine comprises the fat body according to claim 16 and pharmaceutically acceptable excipients.