Microfluidic preparation method for fat body delivery platform

Abstract

The present invention provides a microfluidic preparation method for a fat body delivery platform, comprising the following steps: S1) providing a lipid solution and an aqueous phase solution, wherein the lipid solution comprises a polar lipid and a non-polar lipid, and the molar ratio of the polar lipid to the non-polar lipid is 1:(0.5-6); and S2) introducing each of the lipid solution and the aqueous phase solution into a microfluidic chip to obtain a fat body delivery platform. Compared with the prior art, the present invention relates to rapidly mixing the lipid solution and the aqueous phase solution in a channel of the microfluidic chip to form the fat body delivery platform, which significantly improves the utilization rate of fat body raw materials, can achieve rapid and efficient preparation of fat bodies having high purity and a uniform particle size, and can also achieve loading of fat-soluble small-molecule drugs, thereby paving the way for industrial production of the fat body delivery platform.

Description

Microfluidic preparation method of a fat body delivery platform

[0001] The present application claims priority to the Chinese patent application No. 202411523787.X, filed on October 29, 2024, and entitled "Microfluidic preparation method of a fat body delivery platform", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the field of biological medical technology, and particularly relates to a microfluidic preparation method of a fat body delivery platform. BACKGROUND

[0003] A fat body is an artificially synthesized nanosphere that simulates the lipid structure of lipid droplets organelles in lipoproteins or cells. The main feature of the fat body structure is a nanosphere (30-200 nm) with a neutral lipid core wrapped by a monomolecular phospholipid layer, which is hydrophobic inside and hydrophilic on the surface. The fat body has a significant structural difference from the disclosed liposome nanoparticles, lipid nanoparticle for delivering nucleic acids, and exosome nanoparticles. The hydrophobic neutral lipid core of the fat body has a natural advantage in delivering hydrophobic small molecules, and the peripheral specific monolayer phospholipid membrane simulates the phospholipid membrane structure of lipid droplets or lipoproteins, which can recruit specific proteins and provide a basis for designing targeting molecules.

[0004] The preparation methods of nanoemulsions include high-pressure homogenization, microfluidization, ultrasonication, phase inversion emulsification, and self-emulsification. The basic principle of high-pressure homogenization is to use the high pressure of up to 300 MPa generated in the chamber during the delivery stroke to push the coarse emulsion out of the micron-sized hole of the homogenizer valve, and the effects of turbulence, shear stress, and cavitation make the coarse emulsion into finer droplets. The high-pressure homogenization emulsification process mainly consists of two stages. First, the dispersion phase is broken, resulting in the formation of small droplets in the homogenization chamber, which increases the surface area. Then, the emulsifier molecules can accumulate on the newly formed interface to stabilize the droplets. Repeated high-pressure homogenization can generate a large number of nano-droplets. In the microfluidization method, the lipid raw material is input into a constant-pressure pump system, and then pushed into a fixed-geometry reaction channel at a pressure of up to 30,000 psi (2,068 bar). The lipid raw material can be emulsified into micro-nano emulsion after experiencing consistent high shear rate and impact force in the channel. Compared with the traditional high-pressure homogenization method, the microfluidization method can obtain micro-nano emulsion products with smaller particle size and better particle size distribution uniformity. Ultrasonication uses mechanical vibration and ultrasonic cavitation to prepare nanoemulsions. The strong shock waves generated by sound waves and cavitation form high pressure and turbulence, causing micro-droplets to collapse and break, resulting in small nanoemulsions. However, ultrasonic treatment requires optimal levels of input energy to achieve the smallest droplet diameter. Too high or too low energy will result in poor emulsification. Ultrasonic probes are often used for emulsion droplet preparation of nanoemulsions, and benchtop ultrasonic devices are also used for small-scale production of nanoemulsions. The above are common high-energy input methods for preparing nanoemulsions. Some low-energy input methods are also used to prepare nanoemulsions. Phase inversion emulsification is a method designed to take advantage of the property of some emulsifiers that change their hydrophilicity or lipophilicity with temperature. Some non-ionic surfactants change their spontaneous curvature due to hydration of the polar end, forming oil-in-water emulsions at low temperatures. At high temperatures, the solubility of the emulsifier in water increases, and the oil-in-water emulsion is formed instead. The temperature at which oil-in-water emulsion is converted to oil-in-water emulsion is called the phase inversion temperature. Near the phase inversion temperature, emulsions can be formed, but the emulsions formed at this time are not stable and need to be quickly cooled or heated to generate kinetically stable emulsions with small particle size and narrow particle size distribution. Self-emulsification is also a low-energy input method for emulsion synthesis. This method mixes water, oil, and emulsifier at a specific temperature, and the emulsifier enters the water phase through gentle mechanical stirring, resulting in an increase in oil-water interface area and the formation of nano-droplets.

[0005] However, the above-mentioned emulsion synthesis methods have their own disadvantages. High-pressure homogenization is easy to form amorphous film structure mixed in the emulsion. Phase transfer method and self-emulsification method require specific emulsifiers (surfactants), which do not meet the biological compatibility principle of fat body raw materials. The disclosed fat body synthesis technology mainly includes vortex and ultrasonic methods. The basic principle is to use mechanical force or ultrasonic cavitation to force phospholipids and triglycerides to self-assemble in a water system to form nanoscale particles with a single layer of phospholipid film coating the triglyceride core (Patent No. CN105483076B, US10987431B2, EP3395329A). The vortex method is to introduce phospholipids and neutral fats into a water system (buffer) separately, and high-speed vortex separates large oil droplets into small oil droplets. At the same time, phospholipid molecules can form a monolayer phospholipid film on the surface of small oil droplets. After multiple vortexes, the large droplets are homogenized into small droplets, and then purified to obtain fat bodies. Although the existing vortex technology can obtain high-purity fat bodies, the phospholipid utilization rate is very low (<10%), and the operation process is complex and must be operated manually (using a vortex instrument for 10 seconds of vortex x 24 cycles). Therefore, this technology is suitable for laboratory preparation of small batch research fat bodies, and is not suitable for large-scale production. The ultrasonic method uses mechanical vibration and ultrasonic cavitation generated by an ultrasonic probe to combine turbulent flow to break large neutral fat droplets into small neutral fat droplets, and phospholipid molecules are assembled on the surface of the neutral fat droplets to form fat bodies. However, the current ultrasonic preparation of fat bodies requires probe ultrasonic or ultrasonic cleaning machine water bath ultrasonic emulsification, and the prepared fat bodies also have the problem of low phospholipid utilization rate, which is not suitable for large-scale preparation. SUMMARY

[0006] Therefore, the technical problem to be solved by the present application is to provide a microfluidic preparation method of a fat body delivery platform, which can improve the utilization rate of raw materials, reduce impurity generation, and improve the production efficiency and expand the production scale of fat bodies.

[0007] The present application provides a microfluidic preparation method of a fat body delivery platform, comprising the following steps:

[0008] S1) providing a lipid phase solution and an aqueous phase solution; the lipid phase solution comprises polar lipids and non-polar lipids; the molar ratio of the polar lipids to the non-polar lipids is (10-50):(50-90);

[0009] S2) introducing the lipid phase solution and the aqueous phase solution into a microfluidic chip respectively to obtain a fat body delivery platform.

[0010] Preferably, the solvent of the lipid phase solution is selected from ethanol and / or acetone; the total concentration of the polar lipids and the non-polar lipids in the lipid phase solution is 1-60 mM or 2.5-10 mg / mL.

[0011] Preferably, the polar lipid comprises one or more of phospholipid, cholesterol, functional polar lipid and cationic lipid;

[0012] The phospholipid is selected from one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and sphingomyelin;

[0013] The functional polar lipid is selected from one or more of polyethylene glycol modified sterol, biotin modified sterol, amino acid modified sterol, polypeptide modified sterol, polysaccharide modified sterol, nucleic acid modified sterol, polyethylene glycol modified phospholipid, biotin modified phospholipid, amino acid modified phospholipid, polypeptide modified phospholipid, polysaccharide modified phospholipid and nucleic acid modified phospholipid;

[0014] The cationic lipid is selected from one or more of (2,3-dioleoyl-propyl)-trimethylammonium-chloride salt, (2,3-dioleoyl-propyl)-trimethylamine, 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamine 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;

[0015] The neutral lipid is selected from one or more of triglyceride, wax ester, sterol ester, sterol ester, retinol ester, ether ester, polyhydroxyalkanoate, vitamin A, vitamin D, vitamin E, vitamin K.

[0016] Preferably, the lipid phase solution further comprises polyunsaturated fatty acid; the volume ratio of the non-polar lipid to polyunsaturated fatty acid is 1:(0.5-2).

[0017] Preferably, the polyunsaturated fatty acid can be ω-3 polyunsaturated fatty acid or ω-6 polyunsaturated fatty acid, and specifically comprises one or more of fish oil, corn oil, soybean oil, sunflower seed oil, flaxseed oil and grape seed oil.

[0018] Preferably, the lipid phase solution further comprises drug; the mass ratio of the total mass of the non-polar lipid and polyunsaturated fatty acid to the mass of the drug is (5-20):1.

[0019] Preferably, the aqueous phase solution is selected from water and / or buffer.

[0020] Preferably, the buffer is selected from one or more of phosphate buffer, tris(hydroxymethyl)aminomethane buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and 0.9% NaCl solution.

[0021] Preferably, the total flow rate of the lipid phase solution and the aqueous phase solution in step S2) is 10.5–30 mL / min;

[0022] The flow rate ratio of the lipid phase solution to the aqueous phase solution is 1:1 to 1:20.

[0023] Preferably, the total flow rate of the lipid phase solution and the aqueous phase solution in step S2) is 12-24 mL / min;

[0024] The flow rate ratio of the lipid phase solution to the aqueous phase solution is 1:5.

[0025] Preferably, in step S2), the lipid phase solution and the aqueous phase solution are respectively introduced into the microfluidic chip via an injection pump or a constant flow pump.

[0026] Preferably, the lipid phase solution and the aqueous phase solution are respectively passed into a microfluidic chip to collect the fat body solution, and the fat body solution is concentrated to obtain a fat body delivery platform.

[0027] Preferably, the concentration is ultrafiltration concentration; the molecular weight cutoff of the ultrafiltration concentration is 10-100 kDa.

[0028] The present invention also provides a fat body delivery platform prepared by the above-described microfluidic preparation method.

[0029] Preferably, the average size of the fat body delivery platform is 50–200 nm.

[0030] The present invention also provides an application of the above-mentioned fat body delivery platform in the preparation of drugs or vaccines for the prevention and treatment of diseases.

[0031] Preferably, the disease is cancer.

[0032] The present invention also provides a drug or vaccine comprising the above-described fat body delivery platform and pharmaceutically acceptable excipients.

[0033] This invention provides a microfluidic preparation method for a liposome delivery platform, comprising the following steps: S1) providing a lipid solution and an aqueous solution; the lipid solution includes polar lipids and nonpolar lipids; the molar ratio of the polar lipids to the nonpolar lipids is 1:(0.5-6); S2) passing the lipid solution and the aqueous solution separately into a microfluidic chip to obtain the liposome delivery platform. Compared with the prior art, this invention utilizes the rapid mixing of the lipid solution and the aqueous solution within the channels of the microfluidic chip to form a liposome delivery platform, significantly improving the utilization rate of liposome raw materials. It can rapidly and efficiently prepare high-purity liposomes with uniform particle size, and can also encapsulate lipid-soluble small molecule drugs, paving the way for the industrial production of liposome delivery platforms. Attached Figure Description

[0034] Figure 1 is a microstructure diagram of the fat bodies synthesized by different proportions of dioleoylphosphatidylcholine and trioleic acid glyceride in Example 3 of the present invention.

[0035] Figure 2 shows the structure of the fat bodies synthesized from different proportions of dipalmitoylphosphatidylcholine and trioleic acid glyceride in Example 4 of the present invention after imaging with a structured light illumination microscope or a transmission electron microscope.

[0036] Figure 3 shows the structure of the fat body in Table 4 No. 3 observed by two different microscopic imaging methods in Example 4 of the present invention after imaging with cryo-electron microscopy or transmission electron microscopy.

[0037] Figure 4 shows the particle size analysis and microstructure of the fat bodies synthesized by coating Docetaxel with dipalmitoylphosphatidylcholine and trioleic acid glyceride in Example 9 of the present invention.

[0038] Figure 5 shows the particle size analysis and microstructure of the fat bodies synthesized by loading Docetaxel with a mixture of dipalmitoylphosphatidylcholine and F8 neutral lipids in Example 9 of the present invention.

[0039] Figure 6 is a chromatogram of the fat body drug loading of Docetaxel in Example 9 of the present invention, which was loaded with trioleic acid glyceride and a mixture of fish oil and trioctyl glyceride as neutral lipids. Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0041] This invention provides a microfluidic preparation method for a fat body delivery platform, comprising the following steps: S1) providing a lipid solution and an aqueous solution; the lipid solution includes polar lipids and nonpolar lipids; the molar ratio of the polar lipids to the nonpolar lipids is 1:(0.5-6); S2) passing the lipid solution and the aqueous solution into a microfluidic chip respectively to obtain a fat body delivery platform.

[0042] In this invention, there are no special restrictions on the source of any raw materials; they can be commercially available.

[0043] This invention utilizes a microfluidic chip-based device, such as a microfluidic nanoparticle synthesizer, as the primary equipment for preparing fat bodies. Fat body nanoparticles are rapidly formed by mixing a lipid phase (an organic solvent solution of phospholipids and neutral lipids) and an aqueous phase (buffer solution) within the microchip channels. After concentration and purification, the fat bodies are obtained. The microfluidic preparation equipment uses the E20 pilot-scale machine or self-assembly testing machine manufactured by Hangzhou Tingke Biotechnology Co., Ltd., and the microchip uses a microfluidic mixing unit chip manufactured by both Hangzhou Tingke Biotechnology Co., Ltd. and Zhejiang Yangqing Chip Technology Co., Ltd. This method for preparing a fat body delivery platform significantly improves the utilization rate of fat body raw materials, enabling the rapid and efficient preparation of high-purity fat bodies with uniform particle size. It can also encapsulate lipid-soluble small molecule drugs, paving the way for the industrial production of fat body delivery platforms.

[0044] First, a lipid phase solution and an aqueous phase solution are provided; the lipid phase solution includes polar lipids and nonpolar lipids.

[0045] According to this invention, the polar lipids can be any polar lipids well known to those skilled in the art, without any particular limitation. Preferably, they include, but are not limited to, one or more of phospholipids, cholesterol, functional polar lipids, and cationic lipids. The phospholipids can be natural or synthetic, also known as primary polar lipids, and are preferably phospholipids with a fatty acid chain length of 12 or more carbons. Specifically, they include, but are not limited to, one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin, and sphingomyelin. The functional polar lipids are lipids that help stabilize polar lipid monolayer membranes or provide specific functions, generally natural or synthetic sterols and some polar lipids, including, but not limited to, polyethylene glycol-modified sterols and biotin-modified sterols. The phospholipids include, but are not limited to, one or more of the following: alcohols, 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 include, but are not limited to, (2,3-dioleoyl-propyl)-trimethylammonium chloride, (2,3-dioleoyl-propyl)-trimethylamine, 2,3-dioleoyloxy-N-[2-(speramidocarbamoylamino)ethyl]-N,N-dimethyl-1-propylamine hydrochloride, 1,2-dioleoyl-sn-glycerol-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride.

[0046] 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 more of cholesterol, functional polar lipids, and cationic lipids, and phospholipids; the mass ratio of the sum of the masses of cholesterol, functional polar lipids, and cationic lipids to phospholipids is preferably (0-100):(100-0), more preferably (0-80):(100-20), even more preferably (0-60):(100-40), and even more preferably (0-50):(100-50); in the present invention, the mass ratio of the sum of the masses of cholesterol, functional polar lipids, and cationic lipids to phospholipids can be... The value can be any point within the above range, without any special restrictions. Specifically, it can be 0:100 (i.e., 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 cholesterol, one or more of functional polar lipids and cationic lipids that do not contain phospholipids). The choice can be made according to the needs.

[0047] According to the present invention, the nonpolar lipids are mainly hydrophobic neutral small molecules, including, but not limited to, one or more of triglycerides, wax esters, sterol esters, sterol esters, retinyl esters, ether esters, polyhydroxy fatty acid esters, vitamin A, vitamin D, vitamin E, and vitamin K; wherein, the triglycerides are preferably triglycerides with a fatty acid chain length of 8 or more carbon atoms; the wax esters, sterol esters, sterol esters, or retinyl esters are preferably wax esters, sterol esters, sterol esters, or retinyl esters formed from fatty acids with a carbon chain length of 8 or more 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 methylnaphthoquinone; in some embodiments provided by the present invention, the nonpolar lipids are specifically one or more of trioleic acid glycerides and / or tricaprylic acid glycerides.

[0048] According to the present invention, the molar ratio of polar lipids to nonpolar lipids is (10-50):(50-90). In this invention, the molar ratio of polar lipids to nonpolar lipids can be any value within the above range without special limitations, specifically 10:90, 10:80, 10:70, 10:60, 10:50, 20:80, 20:70, 20:60, 20:50, 22:78, 24:76, 25:75, 29:71, 30:70, 32:68, 35:65, 39:61, 40:60, 41:59, 45:55, or 48:52. Excessively high or low concentrations of polar lipids and nonpolar lipids can lead to membrane fouling or poor fat body homogeneity.

[0049] In one specific embodiment of the present invention, the lipid phase solution comprises polar lipids and nonpolar lipids; the nonpolar lipids comprise neutral lipids; the preferred molar ratio of polar lipids to neutral lipids in the lipid phase solution is (10-50):(50-90); in the present invention, the molar ratio of polar lipids to neutral lipids can be any value within the above range without special limitations, specifically 10:90, 10:80, 10:70, 10:60, 10:50, or 20:80. The ratios are 20:70, 20:60, 20:50, 22:78, 24:76, 25:75, 29:71, 30:70, 32:68, 35:65, 39:61, 40:60, 41:59, 45:55, or 48:52; the neutral lipids are triglycerides with a fatty acid chain length of 8 or more that are well known to those skilled in the art, and there are no special restrictions. In the embodiments provided by this invention, tricaprylic acid glyceride and / or trioleic acid glyceride are specifically used as examples.

[0050] According to the present invention, the solvent of the lipid phase solution is an organic solvent with low physiological toxicity, including but not limited to ethanol and / or acetone.

[0051] According to the present invention, the total concentration of polar lipids and nonpolar lipids in the lipid phase solution is preferably 1-70 mM or 2.5-10 mg / mL; in some embodiments provided by the present invention, the total concentration of polar lipids and nonpolar lipids in the lipid phase solution is specifically 8 mM, 70 mM, 56 mM, 5.9 mM, 5.8 mM, 2.9 mM, 3.1 mM, 3 mM, 4 mM, 2.92 mM, 3.96 mM, 6.55 mM, 7.92 mM, 13.13 mM, 15.83 mM, 45.83 mM, 10 mg / mL, 9 mg / mL, 8 mg / mL, 7 mg / mL, 6 mg / mL, 5 mg / mL, 4 mg / mL, 3 mg / mL, or 2.5 mg / mL.

[0052] According to the present invention, the lipid phase solution can be prepared by any method known to those skilled in the art, and there are no special limitations. In one specific embodiment of the present invention, polar lipids and nonpolar lipids are dissolved in an organic solvent to obtain a lipid phase solution. In another specific embodiment of the present invention, polar lipids and nonpolar lipids are dissolved separately in an organic solvent, and then the solutions of the two are mixed to obtain a lipid phase solution.

[0053] In a specific embodiment of the present invention, the lipid phase solution further includes polyunsaturated fatty acids; the volume ratio of the nonpolar lipids to the polyunsaturated fatty acids is preferably 1:(0.5-2); in the present invention, the volume ratio of the nonpolar lipids to the polyunsaturated fatty acids can be any value within the above range, without any special limitation, specifically 1:0.5, 1:0.8, 1:1, 1:1.2, 1:1.5 or 1:2; the polyunsaturated fatty acids can be polyunsaturated fatty acids well known to those skilled in the art, and can be ω-3 polyunsaturated fatty acids or ω-6 polyunsaturated fatty acids, specifically including one or more of fish oil, corn oil, soybean oil, sunflower seed oil, flaxseed oil and grape seed oil.

[0054] When the lipid phase solution includes polyunsaturated fatty acids, the lipid phase solution can be prepared by dissolving polar lipids, nonpolar lipids, and polyunsaturated fatty acids in an organic solvent to obtain a lipid phase solution; or, mixing and dissolving polar lipids in an organic solvent to obtain a polar lipid stock solution, mixing and dissolving nonpolar lipids and polyunsaturated fatty acids in an organic solvent to obtain a nonpolar lipid and polyunsaturated fatty acid stock solution; and mixing the polar lipid stock solution and the nonpolar lipid and polyunsaturated fatty acid stock solution to obtain a lipid phase solution.

[0055] In another specific embodiment of the present invention, the lipid phase solution further includes a drug; the mass ratio of the total mass of the nonpolar lipids and polyunsaturated fatty acids to the mass of the drug is (5-20):1; in the present invention, the mass ratio of the total mass of the nonpolar lipids and polyunsaturated fatty acids to the mass of the drug can be any value within the above range, without any special limitation, specifically 5:1, 8:1, 10:1, 12:1, 15:1, 18:1 or 20:1; the drug can be any drug that can be encapsulated in fat bodies that is well known to those skilled in the art, without any special limitation, preferably a drug for treating cancer, and in the embodiments provided by the present invention, docetaxel is specifically used as an example for illustration.

[0056] When the lipid phase solution also includes a drug, the lipid phase solution is preferably prepared by the following method: mixing polar lipids with an organic solvent to obtain a polar lipid stock solution; mixing nonpolar lipids, polyunsaturated fatty acids, and the drug to obtain a mixed stock solution; and mixing the polar lipid stock solution with the mixed stock solution to obtain the lipid phase solution. Alternatively, it can be prepared by the following method: mixing polar lipids with an organic solvent to obtain a polar lipid stock solution; mixing nonpolar lipids and polyunsaturated fatty acids with an organic solvent to obtain a nonpolar lipid stock solution; mixing the nonpolar lipid stock solution with the drug to obtain a mixed stock solution; and mixing the polar lipid stock solution with the mixed stock solution until homogeneous to obtain the lipid phase solution.

[0057] According to the present invention, the aqueous phase solution is preferably water and / or a buffer solution; the buffer solution may specifically be one or more of phosphate buffer (PBS), tris(hydroxymethyl)aminomethane buffer (Tris Buffer), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer (HEPES Buffer) and 0.9% NaCl solution (physiological saline).

[0058] The lipid phase solution and aqueous phase solution are respectively introduced into a microfluidic chip to obtain a fat body delivery platform. In this invention, the lipid phase solution and aqueous phase solution are introduced into the microfluidic chip via an injection pump or a constant flow pump. Specifically, the lipid phase solution and aqueous phase solution are preferably separately filled into an injection pump or a container connected to a constant flow pump, and then introduced into the microfluidic chip via the injection pump or constant flow pump. The total flow rate of the lipid phase solution and aqueous phase solution is preferably 10.5-30 mL / min, more preferably 12-24 mL / min. The total flow rate of the lipid phase solution and aqueous phase solution can be any value within the above range without special limitations, specifically 10.5 mL / min, 11 mL / min, 12 mL / min, 12.5 mL / min, 13 mL / min, 15 mL / min, 12 mL / min, 12.5 mL / min, 13 mL / min, 15 mL / min, 16 mL / min, 17 mL / min, 18 mL / min, 19 mL / min, etc. The flow rates are 20 mL / min, 21 mL / min, 22 mL / min, 23 mL / min, 24 mL / min, 25 mL / min, 25.5 mL / min, 26 mL / min, 27 mL / min, 28 mL / min, 29 mL / min, 29.5 mL / min, or 30 mL / min; the preferred flow rate ratio of the lipid phase solution to the aqueous phase solution is 1:1 to 1:20; the flow rate ratio of the lipid phase solution to the aqueous phase solution can be as described above. The values ​​at any point within the range are not subject to any special restrictions, and can specifically be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20; the microfluidic chip can be any chip known to those skilled in the art, and there are no special restrictions. For specific details in this invention, please refer to the published patent of Hangzhou Tingke Biotechnology Co., Ltd. (CN 217093222 U).

[0059] The lipid concentration of liposomes prepared by microfluidic chips is reduced. In a specific embodiment of this invention, the lipid phase solution and aqueous phase solution are respectively passed into the microfluidic chip, and the liposome solution is collected. The liposome solution is then concentrated to obtain a liposome delivery platform. The concentration method can be any concentration method well known to those skilled in the art and is not particularly limited. In this invention, ultrafiltration concentration is preferred. The ultrafiltration concentration device can be an ultrafiltration tube or a tangential flow ultrafiltration system. The molecular weight cutoff of the ultrafiltration concentration is preferably 10-100 kDa, specifically any value between these two points, such as 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or 100 kDa. Ultrafiltration can replace the liquid phase in the product, removing organic solvents and unencapsulated components.

[0060] In another specific embodiment of the present invention, the lipid phase solution and the aqueous phase solution are respectively passed into a microfluidic chip to collect the fat body solution. The fat body solution is then subjected to ultrafiltration concentration, centrifugation, and replenishment of buffer solution. The ultrafiltration concentration and centrifugation process is repeated to obtain a fat body delivery platform. The centrifugation speed is preferably 1,000×g to 5,000×g, more preferably 2,000×g to 4,000×g, even more preferably 2,500×g to 3,500×g, and most preferably 3,000×g. The centrifugation is preferably carried out under low temperature conditions. The temperature of the low temperature conditions is preferably 0℃ to 10℃, more preferably 0℃ to 8℃, even more preferably 2℃ to 6℃, and most preferably 4℃. The centrifugation is preferably carried out until 80% of the liquid phase is filtered out. The number of times the ultrafiltration concentration and centrifugation process is repeated is preferably 1 to 4 times, more preferably 2 to 3 times.

[0061] In this invention, the obtained fat body delivery platform can be analyzed and characterized using optical, chromatographic and other techniques to determine key indicators such as composition and appearance.

[0062] In this invention, the utilization efficiency of phospholipids during the fat body preparation process can be estimated based on the structure of the fat body, and the estimated diameter of the fat body is calculated using the method described in the article. 5,6 The degree of phospholipid excess can be measured by phospholipid redundancy. The calculation formula (1) is as follows:

[0063] Among them, dDetermined and d Estimated These are the measured diameter and estimated diameter of the fat body, respectively.

[0064] The present invention also provides a fat body delivery platform prepared by the above-described microfluidic preparation method.

[0065] Specifically, the average size of the fat body delivery platform is preferably 50-200 nm, more preferably 50-150 nm, even more preferably 70-130 nm, even more preferably 80-120 nm, and most preferably about 100 nm.

[0066] The present invention also provides the application of the fat body delivery platform prepared by the above method in the preparation of drugs or vaccines for the prevention and treatment of diseases.

[0067] Specifically, the disease is cancer, including: breast cancer, lung cancer, kidney cancer, laryngeal cancer, liver cancer, muscle tissue cancer, leukemia, bone cancer, brain cancer, cervical 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, urogenital 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.

[0068] The present invention also provides a drug or vaccine for the prevention and treatment of diseases, comprising pharmaceutically acceptable excipients and a fat body delivery platform prepared by the above method.

[0069] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a microfluidic preparation method for a fat body delivery platform provided by the present invention.

[0070] All reagents used in the following examples are commercially available.

[0071] Example 1

[0072] Process:

[0073] (1) Prepare an ethanolic mixed solution of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and tricaprylin. After complete dissolution, the concentration of DOPC should be 2–10 mM and the concentration of tricaprylin should be 6–60 mM. Use this ethanolic solution as the lipid phase and phosphate-buffered saline (PBS) as the aqueous phase. The specific formulation is: KH2PO4 1.8 mM, Na2HPO4 10.0 mM, KCl 2.7 mM, NaCl 137 mM, pH = 7.4.

[0074] (2) The ethanol phase solution and aqueous phase solution from step (1) are injected into the injection pump, and different flow rate ratios are set. Specifically, the flow rate ratios of ethanol phase to aqueous phase are 1:1, 1:3, 1:5, and 1:6.5, and the total flow rate of lipid phase and aqueous phase is 12 mL / min. The initial product of fat body is collected at the outlet.

[0075] (3) The initial fat body product collected in step (2) is concentrated using an ultrafiltration tube or ultrafiltration device with a molecular weight cutoff of 100 kDa. This involves replacing the liquid phase in the initial product with PBS solution to remove organic solvents. Centrifuge at 3,000 × g and 4 °C until 80% of the liquid phase is removed by filtration. Add an equal volume of PBS to the removed liquid phase and then filter and concentrate again. Repeat this process three times to obtain the fat body product.

[0076] (4) The fat body product obtained in step (3) was measured for particle size distribution and polydispersity index using a dynamic light scattering particle size analyzer.

[0077] Table 1 summarizes the average particle size and polydispersity index (PDI) of fat bodies obtained under different flow rates and concentration ratios. The results show that, for the synthesis of fat bodies from dioleoylphosphatidylcholine and trioctanoic acid glyceride, the optimal flow rate ratio of ethanol to water is 1:5 under a fixed total flow rate of 12 mL / min.

[0078] Table 1. Particle size distribution of fat bodies synthesized from dioleoylphosphatidylcholine and trioctanoic acid glyceride under different flow rates and concentration ratios.

[0079] Example 2

[0080] Process:

[0081] (1) Prepare an ethanolic mixed solution of dioleoylphosphatidylcholine (DOPC) and triolein. After complete dissolution, the concentration of DOPC is 17 mM and the concentration of triolein is 39 mM. Use this ethanolic solution as the lipid phase and phosphate buffered saline (PBS) as the aqueous phase. The specific formulation is: KH2PO4 1.8 mM, Na2HPO4 10.0 mM, KCl 2.7 mM, NaCl 137 mM, pH = 7.4.

[0082] (2) The ethanol phase solution and aqueous phase solution from step (1) are injected into the injection pump respectively, and different flow rate ratios are set. Specifically, the flow rate ratios of ethanol phase to aqueous phase are 1:5, 1:6.5, and 1:20. The total flow rate of lipid phase and aqueous phase is controlled at 10.5-30 mL / min. The initial product of fat body is collected at the outlet.

[0083] (3) The initial fat body product collected in step (2) is concentrated by filtration using an ultrafiltration tube or device with a molecular weight cutoff of 100 kDa. This involves replacing the liquid phase in the initial product with PBS solution to remove organic solvents. Centrifuge at 3,000 × g and 4 °C until 80% of the liquid phase is removed by filtration. Add an equal volume of PBS to the removed liquid phase and then concentrate by filtration. Repeat this process three times to obtain the fat body product.

[0084] (4) The fat body product obtained in step (3) was measured for particle size distribution and polydispersity index using a dynamic light scattering particle size analyzer.

[0085] Table 2 summarizes the average particle size and polydispersity index (PDI) of fat bodies obtained under different flow ratios and concentration ratios. The results indicate that the optimal flow ratio for synthesizing fat bodies from dioleoylphosphatidylcholine and trioleic acid glyceride is 1:5, with a total flow rate of 24 mL / min (self-assembly testing machine).

[0086] Table 2. Particle size distribution of fat bodies synthesized from dioleoylphosphatidylcholine and trioleic acid glyceride under different flow rates and concentration ratios.

[0087] Example 3

[0088] Process:

[0089] (1) Prepare an ethanolic mixed solution of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and triolein. After thorough dissolution, control the total lipid (phospholipid + neutral lipid) concentration to be 2.9–5.9 mM. Use this ethanolic solution as the lipid phase, and phosphate-buffered saline (PBS) as the aqueous phase.

[0090] (2) The ethanol phase solution and aqueous phase solution from step (1) are injected into the injection pump respectively, and different flow rate ratios are set. Specifically, the flow rate ratio of ethanol phase to aqueous phase is 1:5, and the total flow rate of lipid phase and aqueous phase is controlled at 12 mL / min (E20 small test machine). The initial product of fat body is collected at the outlet.

[0091] (3) The initial fat body product collected in step (2) is concentrated by filtration using an ultrafiltration tube or device with a molecular weight cutoff of 100 kDa. This involves replacing the liquid phase in the initial product with PBS solution to remove organic solvents. Centrifuge at 3,000 × g and 4 °C until 80% of the liquid phase is removed by filtration. Add an equal volume of PBS to the removed liquid phase and then concentrate by filtration. Repeat this process three times to obtain the fat body product.

[0092] (4) The fat body product obtained in step (3) was measured with a dynamic light scattering particle size analyzer to measure the particle size distribution and polydispersity index of the fat body, and the micromorphology of the fat body after osmium tetroxide positive staining was observed with a transmission electron microscope.

[0093] Table 3 summarizes the average particle size and polydispersity index (PDI) of fat bodies obtained under different concentration conditions. The predicted diameters of samples 1–5 were 50, 70, 100, 70, and 100 nm, respectively. Calculated according to formula (1), the phospholipid redundancy of the synthetic fat bodies 1–5 were 41.0%, 21.0%, 22.0%, 21.3%, and -7.1%, respectively. Therefore, a total lipid concentration of 2.9 mM and a DOPC / Triolein ratio of 22 / 78 can yield fat bodies close to the theoretical value.

[0094] Table 3. Particle size distribution of fat bodies synthesized from dioleoylphosphatidylcholine and trioleic acid glyceride under different concentrations.

[0095] Figure 1 shows the microstructure of fat bodies synthesized with different ratios of dioleoylphosphatidylcholine and trioleic acid glyceride. The fat bodies were stained with osmium tetroxide. These correspond to samples No. 1(A), No. 2(B), No. 3(C), No. 4(D), and No. 5(E) in Table 3. The scale bars are 1 μm on the left and 200 nm on the right. It can be seen that the neutral lipid core of the fat bodies is uniformly stained black, and the microscopic size distribution of the fat bodies shown by transmission electron microscopy is consistent with the results obtained from dynamic light scattering particle size analysis.

[0096] Example 4

[0097] Process:

[0098] (1) Prepare an ethanolic solution of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and triolein. After thorough dissolution, control the total lipid (phospholipid + neutral lipid) concentration to 3 mM. The molar ratios of dipalmitoyl-sn-glycerocholine and triolein are 41 / 59, 32 / 68, and 24 / 76, respectively. Use this ethanolic solution as the lipid phase, and phosphate-buffered saline (PBS) as the aqueous phase.

[0099] (2) The ethanol phase solution and aqueous phase solution from step (1) are injected into the injection pump respectively, and different flow rate ratios are set. Specifically, the flow rate ratio of ethanol phase to aqueous phase is 1:5, and the total flow rate of lipid phase and aqueous phase is controlled at 12 mL / min (E20 small test machine). The initial product of fat body is collected at the outlet.

[0100] (3) The initial fat body product collected in step (2) is concentrated by filtration using an ultrafiltration tube or device with a molecular weight cutoff of 100 kDa. This involves replacing the liquid phase in the initial product with PBS solution to remove organic solvents. Centrifuge at 3,000 × g and 4 °C until 80% of the liquid phase is removed by filtration. Add an equal volume of PBS to the removed liquid phase and then concentrate by filtration. Repeat this process three times to obtain the fat body product.

[0101] (4) The fat body products obtained in step (3) are measured with a dynamic light scattering particle size analyzer to measure the particle size distribution and polydispersity index of the fat bodies. The micromorphology of the fat bodies stained with LipidTOX Red dye is observed with a structured light illumination super-resolution microscope. The micromorphology of the fat bodies stained with osmium tetroxide positive staining or uranium acetate negative staining is observed with a transmission electron microscope. Alternatively, the micromorphology of the non-destructive fat body samples is observed with a cryo-electron microscope.

[0102] Table 4 summarizes the average particle size and polydispersity index of fat bodies obtained under different molar ratios. The predicted diameters of samples 1-3 are 50, 70, and 100 nm, respectively. According to formula (1), the phospholipid redundancy of the synthesized fat bodies 1-3 are 86.1%, -4.4%, and 7.2%, respectively. Therefore, fat bodies close to the theoretical values ​​can be obtained with a DPPC / Triolein ratio of 32 / 68 or 24 / 76.

[0103] Table 4. Particle size distribution of fat bodies synthesized from dipalmitoylphosphatidylcholine and trioleic acid ester under different molar ratios.

[0104] Figure 2 shows the structures of fat bodies synthesized from different ratios of dipalmitoylphosphatidylcholine and trioleic acid glyceride, as observed by structured light illumination microscopy or transmission electron microscopy. Fluorescence imaging samples were stained with LipidTOX Red (1:1000, excitation wavelength 577 nm, emission wavelength 609 nm). Transmission electron microscopy imaging samples were stained with osmium tetroxide positive staining. These correspond to samples No. 1 (A), No. 2 (B), and No. 3 (C) in Table 4. The scale bars are 2 μm on the left, 1 μm in the middle, and 200 nm on the right. It can be seen that in the fluorescence images, the neutral lipid core of the fat bodies is stained a uniform red; in the positively stained electron microscopy images, the neutral lipid core of the fat bodies is stained a uniform black, showing a black nanospherical structure. In the negatively stained electron microscopy images, the fat bodies exhibit a white nanospherical structure. The microscopic size distribution of the fat bodies shown by structured light illumination microscopy and transmission electron microscopy is consistent with the results obtained from dynamic light scattering particle size analysis.

[0105] Figures 3(A) and (B) show the structures of the fat bodies No. 3 in Table 4 after cryo-electron microscopy (cryo-electron microscopy) or transmission electron microscopy (TEM) imaging, respectively, using two different microscopic imaging methods. The fat bodies were stained with uranium acetate negative staining. (A) corresponds to sample No. 3 in Table 4, with a scale bar of 100 nm for cryo-electron microscopy and (B) for TEM, with a scale bar of 200 nm. Cryo-electron microscopy avoids the potential damage to the sample caused by positive staining in TEM, presenting the original morphology of the fat bodies. Figure 3(A) shows that the cross-section of the fat bodies is a regular circle with a uniform particle size distribution, consistent with the dynamic light scattering results. Figure 3(B) shows the microscopic morphology of the same fat body sample observed under negative staining conditions, which also avoids the potential damage to the sample caused by positive staining, and the results are similar to those of cryo-electron microscopy. Both methods demonstrate that the fat bodies synthesized under these conditions are regular spheres with uniform particle size and stable morphology.

[0106] Example 5

[0107] Process:

[0108] (1) Prepare an ethanol mixture solution of disteaaroyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and triolein. After thorough dissolution, control the total polar lipid (phospholipid + cholesterol) concentration to 1.9 mM, the molar ratio of polar lipids (total concentration of disteaaroyl-sn-glycerocholine and cholesterol) to triolein to 48 / 52, and the triolein concentration to 2.1 mM. The molar concentrations of cholesterol to total polar lipids were 0%, 20%, 40%, 60%, and 80%, respectively. Use this ethanol solution as the lipid phase, and phosphate-buffered saline (PBS) as the aqueous phase.

[0109] (2) The ethanol phase solution and aqueous phase solution from step (1) are injected into the injection pump respectively, and different flow rate ratios are set. Specifically, the flow rate ratio of ethanol phase to aqueous phase is 1:5, and the total flow rate of lipid phase and aqueous phase is controlled at 12 mL / min (E20 small test machine). The initial product of fat body is collected at the outlet.

[0110] (3) The initial fat body product collected in step (2) is concentrated by filtration using an ultrafiltration tube or device with a molecular weight cutoff of 100 kDa. This involves replacing the liquid phase in the initial product with PBS solution to remove organic solvents. Centrifuge at 3,000 × g and 4 °C until 80% of the liquid phase is removed by filtration. Add an equal volume of PBS to the removed liquid phase and then concentrate by filtration. Repeat this process three times to obtain the fat body product.

[0111] (4) The fat body product obtained in step (3) was measured for particle size distribution and polydispersity index using a dynamic light scattering particle size analyzer.

[0112] Table 5 summarizes the average particle size and polydispersity index of fat bodies obtained under different cholesterol molar ratios. The predicted diameter of samples 1-5 is 50 nm. Comparison of the measured diameters of each sample shows that distearylphosphatidylcholine alone cannot synthesize fat bodies (particle size > 1 μm) with triolein via microfluidic control; a certain amount of cholesterol and other auxiliary lipids are required for successful fat body formation. The fat body particle size distribution obtained with a distearylphosphatidylcholine:cholesterol:triolein molar ratio of 29 / 19 / 52 is closest to the predicted value.

[0113] Table 5. Particle size distribution of fat bodies synthesized from distearate, cholesterol, and trioleic acid under different cholesterol molar ratios.

[0114] Example 6

[0115] Process:

[0116] (1) Prepare an ethanolic mixed solution of distearylphosphatidylcholine (DSPC), cholesterol, distearylphosphatidylethanolamine-polyethylene glycol 2000 (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000](ammonium salt), DSPE-PEG2000), and triolein. After thorough dissolution, control the total polar lipid (phospholipid + cholesterol + PEG-modified phospholipid) concentration to 1.9 mM. The molar ratio of polar lipids distearylphosphatidylcholine:cholesterol:distearylphosphatidylethanolamine-polyethylene glycol 2000 was controlled at 60.0 / 38.5 / 1.5. The molar ratios of total polar lipids and triolein were 65 / 35, 48 / 52, and 29 / 71, respectively. Using this ethanol solution as the lipid phase, and phosphate buffered saline (PBS) as the aqueous phase.

[0117] (2) The ethanol phase solution and aqueous phase solution from step (1) are injected into the injection pump respectively, and different flow rate ratios are set. Specifically, the flow rate ratio of ethanol phase to aqueous phase is 1:5, and the total flow rate of lipid phase and aqueous phase is controlled at 12 mL / min (E20 small test machine). The initial product of fat body is collected at the outlet.

[0118] (3) The initial fat body product collected in step (2) is concentrated by filtration using an ultrafiltration tube or device with a molecular weight cutoff of 100 kDa. This involves replacing the liquid phase in the initial product with PBS solution to remove organic solvents. Centrifuge at 3,000 × g and 4 °C until 80% of the liquid phase is removed by filtration. Add an equal volume of PBS to the removed liquid phase and then concentrate by filtration. Repeat this process three times to obtain the fat body product.

[0119] (4) The fat body product obtained in step (3) was measured for particle size distribution and polydispersity index using a dynamic light scattering particle size analyzer.

[0120] Table 6 summarizes the average particle size and polydispersity index of fat bodies obtained under different molar ratios of polar lipids and neutral lipids. The predicted diameter of samples 1-3 is 50 nm. Comparison of the measured diameters of each sample shows that, under the condition of a fixed proportion of polar lipids, the fat body particle size distribution obtained at a molar ratio of polar lipids to triolein of 48 / 52 is closest to the predicted value.

[0121] Table 6. Particle size distribution of synthetic fat bodies containing distearylphosphatidylcholine, cholesterol, distearylphosphatidylethanolamine-polyethylene glycol 2000, and trioleic acid glyceride under different molar ratios of polar and neutral lipids.

[0122] Example 7

[0123] Process:

[0124] (1) Prepare an ethanol mixture solution of distearylphosphatidylcholine (DSPC), cholesterol, distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and triolein. Dissolve the components thoroughly. The molar ratio of polar lipids (DSPC:cholesterol:distearylphosphatidylethanolamine-polyethylene glycol 2000) should be controlled at 60.0 / 38.5 / 1.5. The molar ratio of total polar lipids to triolein should be controlled at 48 / 52. The concentrations of total polar lipids are 1.9 mM, 3.8 mM, 6.3 mM, 7.6 mM, and 22.0 mM, respectively. Use this ethanol solution as the lipid phase and phosphate-buffered saline (PBS) as the aqueous phase.

[0125] (2) The ethanol phase solution and aqueous phase solution from step (1) are injected into the injection pump respectively, and different flow rate ratios are set. Specifically, the flow rate ratio of ethanol phase to aqueous phase is 1:5, and the total flow rate of lipid phase and aqueous phase is controlled at 12 mL / min (E20 small test machine). The initial product of fat body is collected at the outlet.

[0126] (3) The initial fat body product collected in step (2) is concentrated by filtration using an ultrafiltration tube or device with a molecular weight cutoff of 100 kDa. This involves replacing the liquid phase in the initial product with PBS solution to remove organic solvents. Centrifuge at 3,000 × g and 4 °C until 80% of the liquid phase is removed by filtration. Add an equal volume of PBS to the removed liquid phase and then concentrate by filtration. Repeat this process three times to obtain the fat body product.

[0127] (4) The fat body product obtained in step (3) was measured for particle size distribution and polydispersity index using a dynamic light scattering particle size analyzer.

[0128] Table 7 summarizes the average particle size and polydispersity index of fat bodies obtained under different molar ratios of polar and neutral lipids. The predicted diameter of samples 1-3 is 50 nm. Comparison of the measured diameters of each sample shows that under the condition of a fixed polar lipid ratio, a low lipid concentration (polar lipid concentration ≤ 6.3 mM) is beneficial for the synthesis of fat bodies with a diameter close to the predicted diameter.

[0129] Table 7. Particle size distribution of fat bodies synthesized with distearate, cholesterol, distearate phosphatidylcholine-polyethylene glycol 2000, and trioleic acid glyceride under different lipid concentrations.

[0130] Example 8

[0131] Process:

[0132] (1) Prepare stock solutions (12 mM ethanol) for dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso PC). Simultaneously prepare stock solutions (10–50 mM) for tricaprylin, a tricaprylin / fish oil mixture (Tricaprylin:Fish oil = 1:1, v / v, F8), methyl palmitate, and methyl oleate in ethanol or acetone. After thoroughly mixing the different polar and neutral lipid solutions according to the final concentrations in Tables 8 and 9, control the total lipid concentration to 10 mg / mL. Use this ethanol or ethanol-acetone solution as the lipid phase, and phosphate-buffered saline (PBS) as the aqueous phase.

[0133] (2) The lipid phase solution and aqueous phase solution from step (1) are injected into the injection pump respectively, and different flow rate ratios are set. Specifically, the lipid phase: aqueous phase flow rate ratio is 1:5, and the total flow rate of the lipid phase and aqueous phase is controlled at 12 mL / min (E20 small test machine). The initial fat body product is collected at the outlet.

[0134] (3) The initial fat body product collected in step (2) is concentrated by filtration using an ultrafiltration tube or device with a molecular weight cutoff of 100 kDa. This involves replacing the liquid phase in the initial product with PBS solution to remove organic solvents. Centrifuge at 3,000 × g and 4 °C until 80% of the liquid phase is removed by filtration. Add an equal volume of PBS to the removed liquid phase and then concentrate by filtration. Repeat this process three times to obtain the fat body product.

[0135] (4) The fat body product obtained in step (3) was measured for particle size distribution and polydispersity index using a dynamic light scattering particle size analyzer.

[0136] The data in Tables 8 and 9 demonstrate that medium-chain fatty acid triglycerides, long-chain polyunsaturated fatty acid triglycerides, and fatty acid methyl esters can all be synthesized into fat bodies using microfluidics.

[0137] Table 8. Particle size distribution of fat bodies synthesized from dioleoylphosphatidylcholine, medium-chain triglycerides, and long-chain polyunsaturated triglycerides under different lipid concentrations.

[0138] Table 9. Particle size distribution of fat bodies synthesized from distearylphosphatidylcholine, 1-palmitoyl-2-hydroxy-sn-glycerol-3-phosphocholine, and fatty acid methyl esters under different lipid concentrations.

[0139] Example 9

[0140] Process:

[0141] (1) Prepare a stock solution of dipalmitoylphosphatidylcholine (DPPC) (ethanol, 12 mM), and simultaneously prepare an ethanol mixture of triolein and docetaxel (Docetaxel:Triolein = 3:20, m / m; Triolein concentration 10 mM), a mixture of tricaprylin and fish oil (Tricaprylin:Fish oil = 1:1, v / v, F8) and docetaxel (Docetaxel:F8 = 1:10, m / m; F8 concentration 10 mM). Mix the DPPC solution with different neutral lipid solutions thoroughly, controlling the total lipid concentration to 2.5 mg / mL and the molar ratio of polar lipids to neutral lipids to 24:76. Use this ethanol solution as the lipid phase, and phosphate-buffered saline (PBS) as the aqueous phase.

[0142] (2) The lipid phase solution and aqueous phase solution from step (1) are injected into the injection pump respectively, and different flow rate ratios are set. Specifically, the lipid phase: aqueous phase flow rate ratio is 1:5, and the total flow rate of the lipid phase and aqueous phase is controlled at 12 mL / min (E20 small test machine). The initial fat body product is collected at the outlet.

[0143] (3) The initial fat body product collected in step (2) is concentrated by filtration using an ultrafiltration tube or device with a molecular weight cutoff of 100 kDa. This involves replacing the liquid phase in the initial product with PBS solution to remove organic solvents and unencapsulated docetaxel. Centrifuge at 3,000 × g and 4°C until 80% of the liquid phase is removed by filtration. Add an equal volume of PBS to the removed liquid phase and concentrate again. Repeat this process three times to obtain the fat body product.

[0144] The fat body product obtained in step (3) was analyzed for particle size distribution and polydispersity index using a dynamic light scattering particle size analyzer. The microstructure of the fat bodies was observed using transmission electron microscopy or cryo-electron microscopy, and the content of docetaxel encapsulated in the fat bodies was detected by HPLC.

[0145] Figure 4 shows the particle size analysis and microstructure of fat bodies synthesized by coating docetaxel with dipalmitoylphosphatidylcholine and trioleic acid glyceride. (A) Particle size distribution of the fat bodies. Structure observed after transmission electron microscopy: (B) Fat bodies stained with uranium acetate negative staining, scale bar 200 nm. (C) Fat bodies stained with osmium tetroxide positive staining, scale bar 200 nm. Based on the particle size distribution and microstructure of the fat bodies synthesized by DPPC, Triolein, and docetaxel in Figure 4, different electron microscopy observation methods confirmed that the fat bodies are solid spheres, and the particle size distribution is consistent with the results of dynamic light scattering particle size analyzer.

[0146] Figure 5 shows the particle size analysis and microstructure of fat bodies synthesized from DPPC, F8 mixed neutral lipids, and Docetaxel. (A) Particle size distribution of the fat bodies. Structure observed after imaging with transmission electron microscopy or cryo-electron microscopy. (B) Fat bodies stained with osmium tetroxide positive stain, scale bar 200 nm. (C) Fat bodies observed with cryo-electron microscopy, scale bar 100 nm. Figure 5 illustrates the particle size distribution and microstructure of fat bodies synthesized from DPPC, F8 mixed neutral lipids, and Docetaxel. Different electron microscopy observation methods confirm that the fat bodies are solid spheres, and the particle size distribution is consistent with the results of dynamic light scattering particle size analyzer.

[0147] The creation of Docetaxel fat bodies using dipalmitoylphosphatidylcholine, trioleic acid glyceride, and a mixture of F8 neutral lipids was validated, as shown in Figure 6. Figure 6 shows the chromatograms of Docetaxel-loaded fat bodies using trioleic acid glyceride and a mixture of fish oil and caprylic acid glyceride as neutral lipids, respectively. (A) shows the HPLC peak of Docetaxel-loaded fat bodies using trioleic acid glyceride, and (B) shows the peak of Docetaxel-loaded fat bodies using the F8 mixture of neutral lipids. The peak with a retention time of approximately 5.8 minutes is the Docetaxel peak. Calculations show that fat bodies with trioleic acid glyceride as the neutral lipid core are difficult to load with Docetaxel. Fat bodies with a mixture of fish oil and caprylic acid glyceride as the neutral lipid core can load Docetaxel at a concentration of 0.24 mM. Therefore, medium-chain triglycerides and polyunsaturated triglycerides are more advantageous for preparing drug-loaded fat body delivery platforms.

[0148] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A microfluidic preparation method for a fat body delivery platform, characterized in that, Includes the following steps: S1) Provide a lipid phase solution and an aqueous phase solution; the lipid phase solution includes polar lipids and nonpolar lipids; the molar ratio of the polar lipids to the nonpolar lipids is (10-50):(50-90); S2) The lipid phase solution and the aqueous phase solution are respectively introduced into the microfluidic chip to obtain the fat body delivery platform.

2. The microfluidic preparation method according to claim 1, characterized in that, The solvent of the lipid phase solution is selected from ethanol and / or acetone; the total concentration of polar lipids and nonpolar lipids in the lipid phase solution is 1-60 mM or 2.5-10 mg / mL.

3. The microfluidic preparation method according to claim 1, characterized in that, The polar lipids include one or more of phospholipids, cholesterol, functional polar lipids, and cationic lipids; The phospholipids are selected from one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin, and sphingomyelin; The functional polar lipids are selected from one or more of polyethylene glycol modified sterols, biotin modified sterols, amino acid modified sterols, peptide modified sterols, polysaccharide modified sterols, nucleic acid modified sterols, polyethylene glycol modified phospholipids, biotin modified phospholipids, amino acid modified phospholipids, peptide 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-(sperminecarbamoylamino)ethyl]-N,N-dimethyl-1-propylamine hydrochloride, 1,2-dioleoyl-sn-glycerol-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride; The neutral lipids are selected from one or more of the following: triglycerides, wax esters, sterol esters, sterol esters, retinol esters, ether esters, polyhydroxy fatty acid esters, vitamin A, vitamin D, vitamin E, and vitamin K.

4. The microfluidic preparation method according to claim 1, characterized in that, The lipid phase solution also includes polyunsaturated fatty acids; the volume ratio of the nonpolar lipids to the polyunsaturated fatty acids is 1:(0.5-2).

5. The microfluidic preparation method according to claim 4, characterized in that, The lipid phase solution also includes the drug; the total mass ratio of the nonpolar lipids and polyunsaturated fatty acids to the drug is (5-20):

1.

6. The microfluidic preparation method according to claim 1, characterized in that, The aqueous solution is selected from water and / or a buffer solution.

7. The microfluidic preparation method according to claim 6, characterized in that, The buffer solution is selected from one or more of the following: phosphate buffer, tris(hydroxymethyl)aminomethane buffer, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid buffer, and 0.9% NaCl solution.

8. The microfluidic preparation method according to claim 1, characterized in that, The total flow rate of the lipid phase solution and the aqueous phase solution in step S2) is 10.5–30 mL / min; The flow rate ratio of the lipid phase solution to the aqueous phase solution is 1:1 to 1:

20.

9. The microfluidic preparation method according to claim 1, characterized in that, The total flow rate of the lipid phase solution and the aqueous phase solution in step S2) is 12-24 mL / min; The flow rate ratio of the lipid phase solution to the aqueous phase solution is 1:

5.

10. The microfluidic preparation method according to claim 1, characterized in that, In step S2), the lipid phase solution and the aqueous phase solution are respectively introduced into the microfluidic chip through an injection pump or a constant flow pump.

11. The microfluidic preparation method according to claim 1, characterized in that, The lipid phase solution and aqueous phase solution are respectively passed into a microfluidic chip to collect the fat body solution, which is then concentrated to obtain a fat body delivery platform.

12. The microfluidic preparation method according to claim 11, characterized in that, The concentration is ultrafiltration concentration; the molecular weight cutoff of the ultrafiltration concentration is 10-100 kDa.

13. The fat body delivery platform prepared by the microfluidic preparation method according to any one of claims 1 to 12.

14. The fat body delivery platform according to claim 13, characterized in that, The average size of the fat body delivery platform is 50–200 nm.

15. The use of the fat body delivery platform of claim 13 in the preparation of drugs or vaccines for the prevention and treatment of diseases.

16. The application according to claim 15, characterized in that, The disease in question is cancer.

17. A drug or vaccine, characterized in that, The drug or vaccine includes the fat body delivery platform of claim 13 and pharmaceutically acceptable excipients.

Images