Lyophilization method for adiposome

Abstract

Disclosed is a lyophilization method for an adiposome. In the lyophilization process, a lyophilization protective agent is employed to protect the adiposome. The method utilizes lipid materials that are natural or possess high biocompatibility, thereby ensuring effective emulsification during adiposome preparation and enhancing biological safety. The disclosed method establishes a lyophilization process suitable for adiposome-based delivery platforms, in which the adiposome-based delivery platform is preserved in the form of a lyophilized powder. The lyophilized adiposome-based delivery platform is resistant to structural collapse, and upon reconstitution with water, the pre-lyophilization morphology thereof is restored with minimal variations in particle size and polydispersity index. This method enables direct use in related biomedical applications. The lyophilization process improves the storage stability, transportability, and ease of use of the adiposome-based delivery platform.

Description

A freeze-drying method for 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 202410251797.6 and invention name “A method for freeze-drying fat bodies”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of pharmaceutical technology, in particular to a freeze-drying method for fat bodies. Background Art

[0003] Freeze-drying technology is a drying method that freezes water-containing materials below their freezing point, converting the water into ice, and then converting the ice into vapor under a relatively high vacuum. This method can effectively extend the storage time of water-sensitive materials. The freeze-dried material can be restored to its original state after adding water, which can significantly reduce the difficulty of storing and transporting the materials. The main process flow of freeze-drying is: 1) pre-freezing, that is, freezing the sample so that the water in the sample is converted into ice (liquid to solid); 2) primary drying, that is, directly sublimating the ice formed in the pre-freezing step into water vapor under vacuum conditions and removing it from the sample. This process mainly removes free water in the sample; 3) secondary drying, that is, removing the bound water tightly bound to the sample molecules by sublimation under vacuum conditions, and finally obtaining a dry sample. Although freeze-drying is considered a gentle material drying method, it is actually still a potentially destructive process for samples, especially for biological products. Due to the inherent fragility of biological membranes (phospholipid mono- and bilayer membranes) and the environmental sensitivity of biomacromolecules such as proteins, the addition of a lyoprotectant prior to lyophilization is essential to ensure that freeze-dried samples regain their original morphology and bioactivity upon hydration. Commonly used lyoprotectants include polyhydroxy alcohols, sugars, amino acids, polymers, and serum albumin. There are two main hypotheses for their protective mechanism for biomolecules (phospholipid membranes, proteins, etc.): the water-displacement hypothesis and the vitrification hypothesis. The water-displacement hypothesis states that during the drying process, the hydroxyl groups in lyoprotectants, such as sugars, form hydrogen bonds with material molecules such as proteins and phospholipids, replacing the hydrogen bonds originally formed between water and the material, thereby preserving the material molecules' natural conformation. The vitrification hypothesis states that the material molecules are fixed in a glassy lyoprotectant matrix, inhibiting their mobility and reducing degradation, thereby protecting them. The specific mechanism of action of freeze-dried protectants is currently undetermined, but both hypotheses are applicable.

[0004] Fat bodies are artificial nanostructures that mimic lipid droplet organelles / lipoproteins. Their primary lipid structure consists of a hydrophobic neutral lipid core surrounded by a monolayer phospholipid membrane. These structures are synthesized and purified in an aqueous system, resulting in an emulsion. Due to the instability of their primary phospholipid component, fat bodies can undergo phospholipid hydrolysis during prolonged storage, leading to demulsification and deterioration. Therefore, the development of freeze-drying technologies suitable for fat bodies is crucial.

[0005] Given the higher purity of fat bodies and their significantly different lipid composition from lipid delivery vehicles like liposomes and lipid nanoparticles, existing freeze-drying technologies and processes primarily target nanoparticles composed of polar lipids. These technologies are insufficient for achieving the structural integrity of fat bodies, which have a neutral lipid core and a polar lipid membrane, after freeze-drying and reconstitution. Freeze-drying technologies specifically targeting fat bodies require comprehensive consideration of lipid composition, lipid compatibility, and the interaction between lyoprotectants and lipids. Therefore, existing freeze-drying technologies are not suitable for fat bodies, and new freeze-drying technologies suitable for fat bodies are needed.

[0006] Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a freeze-drying method for fat bodies, which can well improve the storage stability of fat bodies.

[0008] To achieve the above object, the present invention provides a freeze-drying method for fat bodies. During the freeze-drying process, a freeze-drying protective agent is used to protect the fat bodies.

[0009] In the present invention, the freeze-drying protective agent can be added during the preparation of the fat body, or during the freeze-drying of the fat body, or added twice during the preparation of the fat body and during the freeze-drying process. Based on this, the freeze-drying method includes the following steps:

[0010] mixing the fat body and a freeze-drying protective agent, and performing vacuum freeze-drying to obtain a fat body freeze-dried powder;

[0011] Alternatively, a freeze-drying protective agent is added during the preparation of the fat body, and the fat body is vacuum-freeze-dried to obtain a fat body freeze-dried powder;

[0012] Alternatively, a freeze-drying protective agent is added during the preparation of the fat body, and after the fat body is obtained, the fat body is mixed with the freeze-drying protective agent and vacuum freeze-dried to obtain the fat body freeze-dried powder.

[0013] The present invention realizes freeze drying of fat bodies. Preferably, the fat bodies used for freeze drying of the present invention include polar lipid membranes and neutral lipids wrapped inside the polar lipid membranes.

[0014] The neutral lipid is preferably one or more of long-chain triglycerides, α-tocopherol, phylloquinone, retinyl acetate, medium-chain triglycerides, long fatty acid methyl esters, long fatty acid ethyl esters, cholesterol esters, soybean oil for injection, fish oil, and corn oil for injection. More preferably, it is tricaprylin, triolein, a mixture of triolein and phylloquinone, a mixture of tricaprylin and fish oil, or a mixture of tricaprylin and soybean oil.

[0015] Optionally, the neutral lipid may be loaded with drugs.

[0016] The present invention has no particular limitation on the above drugs, including but not limited to taxanes and their derivatives such as paclitaxel, docetaxel, camptothecin and its derivatives such as irinotecan, amphotericin B, magnolol, monomethyl auristatin, etc.

[0017] Polar lipids suitable for preparing freeze-dryable fat bodies need to have the following characteristics:

[0018] 1) Ability to emulsify neutral lipids to form fat body nanoparticles and maintain stability;

[0019] 2) Good biocompatibility;

[0020] 3) It has a relatively high phase transition point, which makes it difficult to transform from the gel phase to the liquid crystal phase under common storage temperature conditions, thereby enhancing the fluidity of the polar lipids in the fat body dry powder and causing structural collapse.

[0021] Therefore, the polar lipids used to prepare freeze-dryable fat bodies need to contain: ① a certain amount of long-chain fatty acid phosphatidylcholine, which has good biocompatibility and can maintain the stability of the fat body; ② a certain amount of sterols, which have good biocompatibility and regulate the phase transition temperature of polar lipids to maintain the stability of the fat body; ③ a small amount of DSPE-PEG (2000) amine, which reduces the ability of the fat body surface to adsorb proteins, provides a "stealth" effect for the fat body, and reduces the probability of being cleared by immune cells; ④ optionally contains a small amount of anionic phospholipids, such as phosphatidylglycerol, phosphatidylinositol, etc., to increase the charge density loaded on the fat body surface and increase the dynamic stability of the fat body.

[0022] Preferably, the polar lipid membrane comprises: one or more of long-chain fatty acid phosphatidylcholine, sterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]ammonium salt, and anionic phospholipids.

[0023] Preferably, the polar lipid membrane includes any one of DSPC (distearylphosphatidylcholine), DPPG (dipalmitoylphosphatidylcholine), cholesterol, stigmasterol, sitosterol, phosphatidylglycerol, phosphatidylinositol, DSPE-PEG (2000) amine (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol) -2000] (ammonium salt)), 16:0 Biotinyl Cap PE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotin) (sodium salt)), and egg yolk lecithin or a combination thereof.

[0024] Among them, the molar ratio of the phosphatidylcholine (DSPC, DPPG) preferably accounts for 40% to 100% of the total polar lipids; the molar ratio of the sterols (cholesterol, stigmasterol, sitosterol) preferably accounts for 0 to 58.5% of the total polar lipids; the molar ratio of the phosphatidylglycerol preferably accounts for 0 to 10% of the total polar lipids; the molar ratio of the phosphatidylinositol preferably accounts for 0 to 10% of the total polar lipids; the molar ratio of the DSPE-PEG (2000) amine preferably accounts for 0 to 1.5% of the total polar lipids; and the molar ratio of 16:0 Biotinyl Cap PE preferably accounts for 0 to 60% of the total polar lipids.

[0025] In some specific embodiments of the present invention, the polar lipid membrane comprises DSPC, cholesterol and DSPE-PEG(2000)amine.

[0026] Preferably, the content of DSPC is 60%, the content of cholesterol is 38.5%, and the content of DSPE-PEG (2000) amine is 1.5%.

[0027] In some specific embodiments of the present invention, the polar lipid membrane comprises DSPC, cholesterol, DPPG and DSPE-PEG(2000)amine.

[0028] Preferably, the content of DSPC is 40%-80%, including but not limited to 40%, 60% or 80%, and most preferably 60%.

[0029] Preferably, the content of DPPG is 0-10%, including but not limited to 0, 5% or 10%, and most preferably 10%.

[0030] Preferably, the content of DSPE-PEG(2000)amine is 1.5%.

[0031] The cholesterol content is 100%.

[0032] In some specific embodiments of the present invention, the polar lipid membrane comprises DPPG, cholesterol and DSPE-PEG(2000)amine.

[0033] Preferably, the content of DPPG is 60%, the content of cholesterol is 38.5%, and the content of DSPE-PEG (2000) amine is 1.5%.

[0034] Most preferably, the polar lipid membrane comprises 60% DSPC, 10% DPPG, 1.5% DSPE-PEG (2000) amine and the balance cholesterol.

[0035] The above contents are all molar contents.

[0036] In the fat body, the molar ratio of the total amount of neutral fat to the total amount of polar fat is preferably 3:1 to 5:1, more preferably 4:1.

[0037] The present invention does not have any particular limitation on the preparation method of the fat body, and the method may be a method well known to those skilled in the art. Preferably, the fat body is prepared by the following method:

[0038] S1) evaporating the solvent from the polar lipid solution to obtain a polar lipid membrane;

[0039] S2) mixing the polar lipid membrane and the neutral lipid in a buffer solution to obtain a lipid-water mixture;

[0040] S3) emulsifying the fat-water mixture into crude fat bodies using an ultrasonic homogenizer, and obtaining fat bodies after centrifugal purification.

[0041] The polar lipid solution preferably comprises chloroform, a chloroform-methanol mixed solvent, or a chloroform-methanol-water mixed solvent. In the chloroform-methanol mixed solvent, the volume ratio of chloroform to methanol is preferably 2-5:1-2. In the chloroform-methanol-water mixed solvent, the volume ratio of chloroform to methanol to water is preferably 0.5-3:0.5-3:0.1-1. The polar lipid concentration in the polar lipid solution is preferably 25-32 mM.

[0042] Preferably, the polar lipid solution is added to a container, and the solvent is evaporated by nitrogen flow to obtain a polar lipid film, which is attached to the container wall.

[0043] The polar lipid membrane is then mixed with the neutral lipid in a buffer solution. The buffer solution is preferably a phosphate buffer solution, more preferably PBS; the concentration of the phosphate buffer solution is preferably 5-20 mM, more preferably 20 mM; and the pH of the buffer solution is preferably 5.5-7.4, more preferably 7.4. Preferably, the buffer solution is filtered through a 0.22 μm filter membrane.

[0044] In some specific embodiments of the present invention, the polar lipid comprises DSPC, cholesterol and DSPE-PEG(2000)amine, and the neutral lipid is triolein.

[0045] In some specific embodiments of the present invention, the polar lipid comprises DSPC, dipalmitoylphosphatidylglycerol (DPPG), cholesterol, and DSPE-PEG(2000)amine. The neutral lipid is triolein. Preferably, in the polar lipid solution, the concentration of DSPC is 40% to 100%, the concentration of dipalmitoylphosphatidylglycerol (DPPG) is 0 to 20%, the concentration of DSPE-PEG(2000)amine is 0 to 5%, and the remainder is cholesterol.

[0046] Preferably, in the present invention, the molar ratio of the total amount of the neutral lipids to the total amount of the polar lipids is 3:1 to 5:1, more preferably 4:1.

[0047] In the lipid-water mixture, the mass ratio of total lipids to total water phase is preferably 1:10 to 1:20.

[0048] The frequency of the ultrasonic homogenizer is preferably 20 to 40 kHz, the ultrasonic amplitude is preferably 40% to 80%, the ultrasonic temperature is preferably 15 to 40° C., and the ultrasonic time is preferably 10 to 25 min.

[0049] The centrifugal purification is preferably performed at a temperature of 4 to 10°C, a centrifugal force of 500 to 3000 x g, and a centrifugation time of 1 to 10 minutes. After centrifugation, the lower emulsion, preferably about 80% of the total volume, is collected, and the upper floating large lipid layer, preferably about 20% of the total volume, is removed to obtain the fat bodies.

[0050] Preferably, the purified fat body product prepared above is mixed evenly with an aqueous solution of a freeze-drying protective agent according to a certain proportion, and then subjected to a vacuum freeze-drying treatment.

[0051] The freeze-drying protective agent preferably includes one or more of sucrose, trehalose, glucose, lactose, and mannitol, and more preferably trehalose or sucrose.

[0052] The freeze-drying protective agent used in the present invention is a small polyhydroxy molecule that can provide excellent freeze-drying protective ability; it also has good biocompatibility, low cost, and is easily available.

[0053] The freeze-drying protective agent is preferably prepared in advance as an aqueous solution, and is preferably filtered through a 0.22 μm filter membrane before use.

[0054] The concentration of the freeze-drying protective agent in the system is 0.15M to 1.5M, preferably 0.15M to 0.63M, more preferably 0.17M to 0.51M, and further preferably 0.3M to 0.51M. In some specific embodiments of the present invention, the concentration of the freeze-drying protective agent in the system is 0.15M, 0.30M, 0.51M or 0.63M.

[0055] The concentration of the lyophilization protectant in the fat body system is closely related to the freeze-drying and reconstitution results. A concentration that is too low or too high will not guarantee the freeze-drying effect. Because fat body preparation requires a centrifugation purification step, adding the lyophilization protectant solution after the fat body purification step can prevent it from affecting the centrifugation purification process.

[0056] Optionally, the freeze-drying protective agent further includes: glycine or serine.

[0057] Preferably, the concentration of glycine or serine in the system is ≤0.1M.

[0058] The glycine or serine can be used to protect the targeting peptide and / or protein coated by the outer layer of the fat body during the freeze-drying process.

[0059] Optionally, the present invention may also add a freeze-drying protective agent during the preparation of the fat body.

[0060] The concentration of the freeze-drying protective agent is preferably 5%-15%, more preferably 10%. The ratio of the volume of the freeze-drying protective agent to the total amount of neutral lipids and polar lipids in the fat body is preferably 100 μl: (3-10) mg, more preferably 100 μl: 5 mg.

[0061] The fat bodies prepared after adding the freeze-drying protective agent can be directly subjected to vacuum freeze-drying, or can be mixed with the freeze-drying protective agent again and then subjected to vacuum freeze-drying.

[0062] The vacuum freeze drying is preferably:

[0063] Rapid freezing to -190~-45℃; primary drying temperature -45~-30℃, primary drying time 12~48 hours; secondary drying temperature 20~30℃, secondary drying time 2~6 hours.

[0064] The rapid freezing to -190 to -45°C is preferably performed by maintaining rapid freezing with liquid nitrogen for 5 to 20 minutes and maintaining freezing with a -50°C cold trap for 1 to 3 hours.

[0065] The optimal conditions of the present invention are: the polar lipids include DSPC, dipalmitoylphosphatidylglycerol (DPPG), cholesterol, and DSPE-PEG (2000) amine. The neutral lipid is triolein. In the polar lipid solution, the concentration of DSPC is 60%, the concentration of dipalmitoylphosphatidylglycerol (DPPG) is 10%, the concentration of DSPE-PEG (2000) amine is 1.5%, and the remainder is cholesterol. The PBS pH is 7.4, the PBS concentration is 20mM, and the final sucrose concentration is 0.51M. Under these conditions, the particle size of the reconstituted fat body increases by 12% compared to before lyophilization, and the PDI decreases by 16%.

[0066] Compared with the prior art, the present invention provides a freeze-drying method for fat bodies, comprising the following steps: mixing fat bodies and a freeze-drying protective agent, performing vacuum freeze-drying, and obtaining fat body freeze-dried powder.

[0067] This invention utilizes natural or biocompatible lipid materials to ensure effective emulsification for fat spheroid preparation while enhancing biosafety. Furthermore, a freeze-drying technology has been developed for the fat spheroid delivery platform, enabling storage of the fat spheroid delivery platform as a freeze-dried powder. The freeze-dried fat spheroid delivery platform is resistant to collapse and, upon reconstitution with water, returns to its pre-freeze-dried form with minimal changes in particle size and polydispersity, enabling direct use in relevant biomedical applications. This invention utilizes freeze-drying technology to enhance the storage stability, transportation, and ease of use of the fat spheroid delivery platform. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG1 is a graph showing the effect of different sucrose concentrations on the freeze-drying effect of fat bodies;

[0069] FIG2 is a comparison of the average particle size and polydispersity coefficient of fat bodies prepared under different conditions before and after freeze-drying;

[0070] Figure 3 shows the changes in average particle size and polydispersity coefficient of freeze-dried fat bodies after storage at 4°C;

[0071] Figure 4 shows the transmission electron microscopic morphology of freeze-dried fat bodies after storage at 4°C;

[0072] Figure 5 shows the changes in average particle size and polydispersity coefficient of freeze-dried fat bodies after storage at 4°C;

[0073] FIG6 is a comparison of the particle size and morphology of lyophilized and non-lyophilized docetaxel fat bodies;

[0074] FIG7 is a graph showing the particle size distribution and docetaxel concentration curves of freeze-dried docetaxel fat bodies under different conditions. DETAILED DESCRIPTION

[0075] To further illustrate the present invention, the freeze-drying method of the fat body provided by the present invention is described in detail below with reference to the examples.

[0076] Example 1

[0077] Process:

[0078] (1) Preparation of the fat body preparation system: 32 mM DSPC, 32 mM cholesterol and 32 mM DSPE-PEG (2000) amine solutions were mixed in proportion to form a polar lipid component (the specific mixing ratio was DSPC 60%, cholesterol 38.5%, DSPE-PEG (2000) amine 1.5%, mol%), the organic solvent was evaporated under a nitrogen flow, and then phosphate buffer (0.137 M NaCl, 0.0027 M KCl, 0.01 M Na2HPO4 and 0.0018 M KH2PO4) and a neutral lipid component (100% triolein) were added.

[0079] (2) Preparation of Fat Bodies: The lipid-water mixture from step (1) was emulsified using an ultrasonic homogenizer at a frequency of 40 kHz, an amplitude of 60%, and a constant temperature of 25 ± 5°C for 15 minutes to form crude fat bodies. The crude fat bodies were then separated into purified fat bodies by centrifugation at 4°C, 1000 × g, and 5 minutes. The lower 80% volume of the emulsion was taken as the purified fat body.

[0080] (3) The purified fat bodies obtained in step (2) 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.

[0081] (4) The purified fat body product obtained in step (3) was mixed evenly with a sucrose aqueous solution (filtered through a 0.22 μm filter membrane) in proportion, with the concentrations of the sucrose aqueous solution being 0.15, 0.30, and 0.63 M, respectively, with no sucrose aqueous solution added as a control.

[0082] (5) The mixed emulsion obtained in step (4) was rapidly frozen to -190°C with liquid nitrogen and held for 5 minutes, then transferred to a vacuum freeze dryer for freeze drying. The temperature was raised to -35 to -30°C and held for 1 hour. The primary drying temperature was -35 to -30°C, the primary drying time was 20 hours, and the secondary drying temperature was 20 to 25°C, the secondary drying time was 4 hours. The sample was removed and sealed for storage.

[0083] The sample obtained in step (5) was reconstituted with water and its microscopic morphology was observed using an optical microscope, and its average particle size and polydispersity were measured using a dynamic light scattering particle size analyzer.

[0084] Figure 1 shows the effect of different sucrose concentrations on the freeze-drying effect of fat bodies. Figure A shows the fat body before freeze-drying, Figure B shows the fat body after freeze-drying and reconstruction, Figure C shows the fat body after freeze-drying and reconstruction containing 0.15M sucrose, Figure D shows the fat body after freeze-drying and reconstruction containing 0.30M sucrose, and Figure E shows the microscopic morphology of the fat body after freeze-drying and reconstruction containing 0.63M sucrose; Figure F shows the comparison of the average particle size (left) and polydispersity index (right) of the fat body before and after freeze-drying and reconstruction.

[0085] Figure 1 shows the differences in micromorphology, particle size, and polydispersity index after reconstitution of fat bodies after direct freeze-drying and freeze-drying with sucrose solution. As shown, adding different concentrations of sucrose solution as a freeze-drying protectant can restore the fat bodies to their unfreeze-dried state after reconstitution with water. In contrast, the absence of sucrose completely destroys the fat body structure, preventing proper reconstitution to its pre-freeze-dried state. Furthermore, reconstitution is most effective when the sucrose concentration is ≥0.30 M.

[0086] Example 2

[0087] Process:

[0088] (1) Preparation of the fat body preparation system: DSPC, cholesterol, dipalmitoylphosphatidylglycerol (DPPG) and DSPE-PEG (2000) amine solutions were mixed in proportion to form a polar lipid component (see Table 3, No. 12 for the specific mixing ratio), the organic solvent was evaporated under a nitrogen flow, and then phosphate buffer and a neutral lipid component (100% triolein) were added.

[0089] (2) Preparation of Fat Bodies: The lipid-water mixture from step (1) was emulsified using an ultrasonic homogenizer at a frequency of 40 kHz, an amplitude of 60%, and a constant temperature of 25 ± 5°C for 15 minutes to form crude fat bodies. The crude fat bodies were then separated into purified fat bodies by centrifugation at 4°C, 1000 × g, and 5 minutes. The lower 80% volume of the emulsion was taken as the purified fat body.

[0090] (3) The purified fat bodies obtained in step (2) 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.

[0091] (4) The purified fat body product obtained in step (3) was mixed uniformly with aqueous solutions of lyophilization protectants (filtered through a 0.22 μm filter) of varying types and concentrations in proportion. Table 1 shows the mixing of different types of lyophilization protectants with fat bodies at a final concentration of 0.3 M. Table 2 shows the simultaneous mixing of glucose or sucrose at a final concentration of 0.3 M with fat bodies, respectively, and varying concentrations of glycine or serine.

[0092] (5) The mixed emulsion obtained in step (4) was rapidly frozen to -190°C with liquid nitrogen and held for 5 minutes, then transferred to a vacuum freeze dryer for freeze drying. The temperature was raised to -35 to -30°C and held for 1 hour. The primary drying temperature was -35 to -30°C, the primary drying time was 20 hours, and the secondary drying temperature was 20 to 25°C, the secondary drying time was 4 hours. The sample was removed and sealed for storage.

[0093] (6) After the sample obtained in step (5) was reconstituted with water, its microscopic morphology was observed using an optical microscope, and its average particle size and polydispersity were measured using a dynamic light scattering particle size analyzer.

[0094] The results are shown in Table 1:

[0095] Table 1. Comparison of average particle size and polydispersity index of fat bodies before and after freeze-drying

[0096] Note: The final concentration of the freeze-drying protectant in the fat body emulsion before lyophilization was 0.3 M. Mean = ± SEM, n ≥ 3.

[0097] Table 2. Comparison of average particle size and polydispersity index of fat bodies before and after freeze-drying

[0098] Note: N / A indicates that the emulsion could not be reconstituted after freeze-drying. The final concentration of the freeze-drying protectant used is indicated in the table. Mean = ± SEM, n ≥ 3.

[0099] Tables 1 and 2 show the results of freeze-drying and reconstitution of fat bodies in the presence of different lyoprotectants or combinations. Glucose, lactose, mannitol, sucrose, and trehalose all serve as effective lyoprotectants for fat bodies. Amino acid lyoprotectants, such as glycine and serine, alone do not effectively reconstitute fat bodies after freeze-drying. Low concentrations (≤0.1 M) of glycine or serine can be combined with sucrose to achieve a protective effect on freeze-dried fat bodies.

[0100] Example 3

[0101] Process:

[0102] (1) Preparation of the fat body preparation system: DSPC, cholesterol, dipalmitoylphosphatidylglycerol (DPPG) and DSPE-PEG (2000) amine solutions were mixed in proportion to form a polar lipid component (see Table 3 for the specific mixing ratios). The organic solvent was evaporated under a nitrogen flow, and then phosphate buffer and a neutral lipid component (100% triolein) were added.

[0103] (2) Preparation of Fat Bodies: The lipid-water mixture from step (1) was emulsified using an ultrasonic homogenizer at a frequency of 40 kHz, an amplitude of 60%, and a constant temperature of 25 ± 5°C for 15 minutes to form crude fat bodies. The crude fat bodies were then separated into purified fat bodies by centrifugation at 4°C, 1000 × g, and 5 minutes. The lower 80% volume of the emulsion was taken as the purified fat body.

[0104] (3) The purified fat bodies obtained in step (2) 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.

[0105] (4) The purified fat body product obtained in step (3) is mixed evenly with a freeze-drying protective agent aqueous solution (filtered with a 0.22 μm filter membrane) in proportion.

[0106] (5) The mixed emulsion obtained in step (4) was rapidly frozen to -190°C with liquid nitrogen and held for 5 minutes, then transferred to a vacuum freeze dryer for freeze drying. The temperature was raised to -35 to -30°C and held for 1 hour. The primary drying temperature was -35 to -30°C, the primary drying time was 20 hours, and the secondary drying temperature was 20 to 25°C, the secondary drying time was 4 hours. The sample was removed and sealed for storage.

[0107] (6) After the sample obtained in step (5) was reconstituted with water, its microscopic morphology was observed using an optical microscope, and its average particle size and polydispersity were measured using a dynamic light scattering particle size analyzer.

[0108] Table 3. Different experimental conditions for fat body freeze-drying

[0109] Figure 2 compares the average particle size and polydispersity index of fat bodies prepared under different conditions before and after freeze-drying and reconstitution. Panel A shows the change in average particle size of fat bodies before and after freeze-drying and reconstitution (mean = ± SEM, n ≥ 3); Panel B shows the change in average polydispersity index (PDI) of fat bodies before and after freeze-drying and reconstitution (n ≥ 3).

[0110] Table 3 shows the experimental setup for different conditions. Five fundamental influencing factors are the molar ratio of zwitterionic phospholipids to total polar lipids (mol%), i.e., zwitterionic phospholipids (in this experiment, DSPC is the zwitterionic phospholipid); the molar ratio of anionic phospholipids to total polar lipids (mol%), i.e., DPPG (in this experiment, DPPG is the anionic phospholipid); PBS pH; PBS concentration; and cryoprotectant concentration (in this experiment, sucrose is the cryoprotectant). All fat body formulations in this experiment contained 1.5% DSPE-PEG(2000)amine. After DSPC, the remainder of DPPG and 1.5% DSPE-PEG(2000)amine was supplemented with cholesterol. The results show that the optimal lyophilization reconstitution effect is achieved when the DSPC concentration is 60%, the DPPG concentration is 10%, the PBS pH is 7.4, the PBS concentration is 20 mM, and the final sucrose concentration is 0.51 M. After reconstitution, the particle size of the fat body increased by 12% and the PDI decreased by 16% compared with that before freeze-drying.

[0111] Example 4

[0112] Process:

[0113] (1) Preparation of the liposome preparation system: DSPC, cholesterol, and DSPE-PEG(2000)amine solutions were mixed in the appropriate ratio to form a polar lipid component (DSPC:cholesterol:DSPE-PEG(2000)=60:38.5:1.5, mol / mol / mol). The organic solvent was evaporated under a nitrogen stream, and phosphate buffer was added. Triolein and phylloquinone were mixed and dissolved at room temperature as a neutral lipid component. The neutral lipid was transferred to the polar lipid-water mixture at a lipid-water ratio of 1:20 (v / v).

[0114] (2) Preparation of Fat Bodies: The lipid-water mixture from step (1) was emulsified using an ultrasonic homogenizer at an amplitude of 60% and a constant temperature of 25±5°C for 15 minutes to form crude fat bodies. The crude fat bodies were then separated into purified fat bodies by centrifugation at 4°C, 1000×g, and 5 minutes. The lower 80% volume of the emulsion was taken as the purified fat body.

[0115] (3) The purified fat body product obtained in step (2) was mixed evenly with a sucrose aqueous solution (filtered with a 0.22 μm filter membrane, with a final concentration of sucrose in the fat body mixture of 0.34 M) in proportion, and the resulting fat body emulsion was divided into equal volumes of 60 μl each for freeze-drying.

[0116] (4) The mixed emulsion obtained in step (3) was rapidly frozen to -190°C with liquid nitrogen and held for 5 minutes, then transferred to a vacuum freeze dryer for freeze drying. The temperature was raised to -35 to -30°C and held for 1 hour. The primary drying temperature was -35 to -30°C, the primary drying time was 20 hours, and the secondary drying temperature was 20 to 25°C, the secondary drying time was 4 hours. The sample was removed and stored.

[0117] (5) The freeze-dried fat bodies obtained in step (4) were sealed and stored in the dark at 4°C. 55 μl of water was added over time to restore the emulsion. The average particle size distribution was measured. The micromorphology of the fat bodies before and after freeze-drying was observed and compared using a transmission electron microscope with positive staining. The same test was also applied to fat bodies stored in an emulsion.

[0118] Figure 3 shows the changes in average particle size and polydispersity index of freeze-dried fat bodies after storage at 4°C. DSPC C LY represents freeze-dried fat bodies. Panel A shows the change in average particle size over time for freeze-dried fat bodies (mean = ±SD, n ≥ 3); Panel B shows the change in polydispersity index over time for freeze-dried fat bodies (n ≥ 3).

[0119] Figure 4 shows the transmission electron microscopic morphology of freeze-dried fat bodies after storage at 4°C. A shows the microscopic morphology of freshly prepared fat bodies; B shows the microscopic morphology of freeze-dried fat bodies after reconstitution at 4°C for 90 days. Scale bar = 500 nm.

[0120] Example 5

[0121] Process:

[0122] (1) Preparation of the liposome preparation system: DPPG, cholesterol, and DSPE-PEG (2000) amine solutions were mixed in the appropriate ratio to form a polar lipid component (DPPG: cholesterol: DSPE-PEG (2000) = 60:38.5:1.5, mol / mol / mol). The organic solvent was evaporated under a nitrogen stream, and phosphate buffer was added. Triolein and phylloquinone were mixed and dissolved at room temperature as a neutral lipid component. The neutral lipid was transferred into the polar lipid-water mixture at a lipid-water ratio of 1:20 (v / v).

[0123] (2) Preparation of Fat Bodies: The lipid-water mixture from step (1) was emulsified using an ultrasonic homogenizer at a frequency of 40 kHz, an amplitude of 60%, and a constant temperature of 25 ± 5°C for 15 minutes to form crude fat bodies. The crude fat bodies were then separated into purified fat bodies by centrifugation at 4°C, 1000 × g, and 5 minutes. The lower 80% volume of the emulsion was taken as the purified fat body.

[0124] (3) The purified fat body product obtained in step (2) was mixed evenly with a sucrose aqueous solution (filtered with a 0.22 μm filter membrane, with a final concentration of sucrose in the fat body mixture of 0.34 M) in proportion, and the resulting fat body emulsion was divided into equal volumes of 60 μl each for freeze-drying.

[0125] (4) The mixed emulsion obtained in step (3) was rapidly frozen to -190°C with liquid nitrogen and held for 5 minutes, then transferred to a vacuum freeze dryer for freeze drying. The temperature was raised to -35 to -30°C and held for 1 hour. The primary drying temperature was -35 to -30°C, the primary drying time was 20 hours, and the secondary drying temperature was 20 to 25°C, the secondary drying time was 4 hours. The sample was removed and stored.

[0126] (5) The freeze-dried fat bodies obtained in step (4) were sealed and stored in the dark at 4°C. 55 μl of water was added over time to restore the emulsion state. The average particle size distribution was measured.

[0127] Figure 5 shows the changes in average particle size and polydispersity index of freeze-dried fat bodies after storage at 4°C. DPPC C LY represents freeze-dried fat bodies. Panel A shows the change in average particle size over time for freeze-dried fat bodies (mean = ±SD, n ≥ 3); Panel B shows the change in polydispersity index over time for freeze-dried fat bodies (n ≥ 3).

[0128] Example 6

[0129] Process:

[0130] (1) Preparation of the fat body preparation system: Prepare an egg yolk lecithin Egg PC ethanol solution (32 mM) and a docetaxel ethanol solution (100 mM). Mix egg yolk lecithin Egg PC, tricaprylin, fish oil, and docetaxel in proportion to form a lipid component solution (Egg PC 17%, tricaprylin 41%, fish oil 41%, docetaxel 1%, m%). Add 5 mg of the mixed lipid solution to a 1.5 ml EP tube, and evaporate the ethanol under a gentle nitrogen flow. Add 100 μl of a 10% sucrose aqueous solution (filtered through a 0.22 μm filter membrane) to the mixed lipid solution to form a lipid-water mixture.

[0131] (2) Preparation of Fat Bodies: The lipid-water mixture from step (1) was emulsified using an ultrasonic homogenizer at a frequency of 40 kHz, an amplitude of 60%, and a constant temperature of 25±5°C for 15 minutes to form crude fat bodies. The crude fat bodies were then separated into purified fat bodies by centrifugation at 4°C, 1000×g, and 5 minutes. 80% of the volume of the emulsion layer was removed to obtain the purified docetaxel fat bodies.

[0132] (3) The purified docetaxel fat body product obtained in step (2) was divided into equal volumes of 60 μl each, frozen in a cold trap at -45 to -85°C for 30 to 60 minutes, and transferred to a freeze dryer for freeze drying.

[0133] (4) The frozen sample obtained in step (3) was heated to -35 to -30°C in a freeze dryer and maintained for 1 hour. The primary drying temperature was -35 to -30°C for 20 hours and the secondary drying temperature was 20 to 25°C for 4 hours. The sample was removed and stored.

[0134] (5) The freeze-dried fat bodies obtained in step (4) were sealed and stored in the dark at 4°C. 55 μl of water was added to restore the emulsion state before testing, and the average particle size distribution was measured. The micromorphology of the fat bodies before and after freeze-drying was observed and compared using a fluorescence microscope.

[0135] Figure 6 compares the particle size and morphology of freeze-dried and non-freeze-dried docetaxel fat bodies. A shows the particle size distribution of freshly prepared docetaxel fat bodies, with an average particle size of 125.8 ± 12.1 nm and a PDI of 0.123. B shows the particle size distribution of docetaxel fat bodies recovered after freeze-drying, with an average particle size of 130.3 ± 17.8 nm and a PDI of 0.171. C and D show the microstructures of freshly prepared fat bodies and fat bodies recovered after freeze-drying, respectively, using Nile Red as the staining agent. E shows the appearance of freshly prepared fat bodies (control), freeze-dried fat body powder, and fat bodies recovered after freeze-drying. This demonstrates that docetaxel fat bodies can be freeze-dried and reconstituted using this method without affecting their appearance or microscopic morphology.

[0136] Example 7

[0137] Process:

[0138] (1) Fat body preparation system: Egg yolk lecithin PC-98T, tricaprylin, soybean oil, and docetaxel were mixed in appropriate proportions to form a lipid component (PC-98T 19%, tricaprylin 40%, soybean oil 40%, docetaxel 1%, m%) and dissolved at 60-70°C to form the lipid phase. A 10% sucrose aqueous solution was filtered through a 0.22 μm filter membrane to form the aqueous phase.

[0139] (2) Preparation of fat bodies: The lipid phase and aqueous phase in step (1) are transferred into different screw-mouth syringes (10-60 ml) respectively. The screw-mouth syringe is fixed on a horizontal push pump, and the lipid-water flow rate ratio is set to 1:9, and the total flow rate is 2 ml / min. The mixing channel is fixed in a water bath ultrasonic pool, and the ultrasonic parameters are set to a frequency of 40 kHz, a temperature of 50-60°C, an amplitude of 60%, and a residence time of the lipid-water mixture in the mixing channel of 5.5 min. The primary emulsion obtained by ultrasound is separated into purified fat bodies by centrifugation purification method, with a centrifugation temperature of 4°C, 1000×g, and a centrifugation time of 5 minutes. Take 80% of the volume of the emulsion layer and pass it through a 0.22 μm filter membrane to obtain the purified docetaxel fat bodies.

[0140] (3) The purified docetaxel fat body product obtained in step (2) is divided into 1.5 ml EP tubes or 1.5 ml glass vials at a rate of 200 μl each. The product is frozen in a -45 to -85°C cold trap for 30 to 60 minutes, and then transferred to a freeze dryer for freeze drying. Simultaneously, the purified docetaxel fat body product is mixed evenly with 200 μl of a 0.9 M sucrose aqueous solution (filtered through a 0.22 μm membrane) and divided into 1.5 ml EP tubes or 1.5 ml glass vials. The product is frozen in a -45 to -85°C cold trap for 30 to 60 minutes, and then transferred to a freeze dryer for freeze drying.

[0141] (4) The frozen sample obtained in step (3) was heated to -35 to -30°C in a freeze dryer and maintained for 1 hour. The primary drying temperature was -35 to -30°C for 20 hours and the secondary drying temperature was 20 to 25°C for 4 hours. The sample was removed and stored.

[0142] (5) The freeze-dried docetaxel fat bodies obtained in step (4) were sealed and stored in the dark at 4°C. Water was added according to different recovery volumes to restore the emulsion state. The average particle size distribution was measured. The micromorphology of the reconstituted fat bodies was observed using a fluorescence microscope.

[0143] Figure 7 shows the particle size distribution and docetaxel concentration curves of lyophilized docetaxel fat bodies under different conditions. a 100, a 90, a 80, a 70, a 60, and a 50 represent the reconstituted fat bodies after lyophilizing 200 μl of fat body and adding 180 μl of water, 160 μl of water, 140 μl of water, 120 μl of water, 100 μl of water, and 80 μl of water, respectively. b100, b90, b80, b70, b60, b50, b40, and b30 were mixed with 200 μl of 0.9 M sucrose aqueous solution and lyophilized, followed by reconstitution with 360 μl, 320 μl, 280 μl, 240 μl, 200 μl, 160 μl, 120 μl, and 80 μl of water, respectively. Origin represents the fat body before lyophilization. A represents the average particle size of the fat body after lyophilization recovery in each group, B represents the polydispersity index (PDI) of the fat body after lyophilization recovery in each group, and C represents the docetaxel (DTX) concentration of the fat body after lyophilization recovery in each group.

[0144] The above embodiments are only intended to help understand the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by those skilled in the art, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A freeze-drying method for fat bodies, characterized in that: During the freeze-drying process, a freeze-drying protectant is used to protect the fat bodies.

2. The freeze-drying method according to claim 1, wherein The following steps are involved: mixing the fat body and a freeze-drying protective agent, and performing vacuum freeze-drying to obtain a fat body freeze-dried powder; Alternatively, a freeze-drying protective agent is added during the preparation of the fat body, and the fat body is vacuum-freeze-dried to obtain a fat body freeze-dried powder; Alternatively, a freeze-drying protective agent is added during the preparation of the fat body, and after the fat body is obtained, the fat body is mixed with the freeze-drying protective agent and vacuum freeze-dried to obtain the fat body freeze-dried powder.

3. The freeze-drying method according to claim 1, wherein The freeze-drying protective agent includes one or more of sucrose, trehalose, glucose, lactose and mannitol.

4. The freeze-drying method according to claim 3, wherein The freeze-drying protective agent further comprises: glycine or serine; The concentration of glycine or serine in the system is ≤0.1M.

5. The freeze-drying method according to claim 1, wherein The concentration of the freeze-drying protective agent in the system is 0.15M to 1.5M; The concentration is preferably 0.15M to 0.63M; The concentration is more preferably 0.17M to 0.51M; The concentration is further preferably 0.3M to 0.51M; Most preferably, the concentration is 0.15M, 0.30M, 0.51M or 0.63M.

6. The freeze-drying method according to claim 1, wherein The vacuum freeze drying is: Rapid freezing to -190~-45℃; primary drying temperature -45~-30℃, primary drying time 12~48 hours; secondary drying temperature 20~30℃, secondary drying time 2~6 hours.

7. The freeze-drying method according to claim 1, wherein The fat body includes a polar lipid membrane and neutral lipids wrapped in the polar lipid membrane.

8. The freeze-drying method according to claim 7, wherein The neutral lipid is selected from one or more of long-chain triglycerides, α-tocopherol, phylloquinone, retinol acetate, medium-chain triglycerides, long fatty acid methyl esters, long fatty acid ethyl esters, cholesterol esters, soybean oil for injection, fish oil, and corn oil for injection; The polar lipid membrane comprises one or more of long-chain fatty acid phosphatidylcholine, sterol, 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]ammonium salt, and anionic phospholipids.

9. The freeze-drying method according to claim 8, wherein The polar lipid membrane comprises: One or more of distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, cholesterol, stigmasterol, sitosterol, phosphatidylglycerol, phosphatidylinositol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]ammonium salt, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotin) sodium salt, and egg yolk lecithin.

10. The freeze-drying method according to claim 7, wherein The fat body is prepared according to the following method: S1) evaporating the solvent from the polar lipid solution to obtain a polar lipid membrane; S2) mixing the polar lipid membrane and the neutral lipid in a buffer solution to obtain a lipid-water mixture; S3) emulsifying the fat-water mixture into crude fat bodies using an ultrasonic homogenizer, and obtaining fat bodies after centrifugal purification.

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