Docetaxel adiposome formulation, preparationmethod therefor, and use thereof

Abstract

Provided are a docetaxel adiposome formulation, a preparation method therefor, and use thereof. The docetaxel adiposome formulation comprises a monomolecular phospholipid membrane and docetaxel and a neutral lipid that are wrapped in the monomolecular phospholipid membrane. Targeted modification is further carried out on the surface of the docetaxel adiposome. The adiposome formulation has good bioavailability and safety, and the anti-cancer effect of the formulation is significantly superior to that of a clinically used docetaxel injection.

Description

Docetaxel fat body preparation and its preparation method and application

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on March 5, 2024, with application number 202410251160.7 and invention name “Docetaxel Fat Body Formulation, Preparation Method and Application Thereof”, 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 docetaxel fat body preparation and a preparation method and application thereof. Background Art

[0003] Docetaxel is a chemotherapy drug used to treat various types of cancer, including breast cancer, non-small cell lung cancer, prostate cancer, gastric cancer, and head and neck cancer. It binds to microtubules, preventing their depolymerization, thereby inhibiting cell division and leading to cell death. Docetaxel is a hydrophobic drug with a logP (oil-water partition coefficient) of 1.6 and an extremely low solubility in water of only 0.006 mg / mL. Docetaxel currently used clinically is in the form of an injection. To improve its solubility, polysorbate 80 (Tween 80) and ethanol are often added. However, these can lead to serious adverse reactions, such as liver dysfunction, allergic reactions, and neurotoxicity [van Zuylen L, Verweij J, Sparreboom A. Role of formulation vehicles in taxane pharmacology. Invest New Drugs 2001; 19: 125-141.; Engels FK, Mathot RA, Verweij J. Alternative drug formulations of docetaxel: a review. Anticancer Drugs 2007; 18: 95-103.]. Consequently, its hydrophobicity significantly affects its absorption and bioavailability, while also leading to varying degrees of toxic side effects, severely limiting its clinical application.

[0004] To solve the above problems, nanocarriers are widely used in the development of new docetaxel formulations. The following are commonly used methods: 1) Liposomes [Immordino ML, Brusa P, Arpicco S et al. Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing docetaxel. J Control Release 2003; 91: 417-429.; Chen Y, Chen J, Cheng Y et al. A lyophilized sterically stabilized liposo me-containing docetaxel: in vitro and in vivo evaluation. J Liposome Res 2017; 27: 64-73.; Zhai G, Wu J, Xiang G et al. Preparation, characterization and pharmacokinetics of folate receptor-targeted liposomes for docetaxel delivery. J Nanosci Nanotechnol 2009;9:2155-2161.] Liposomes are vesicles composed of a phospholipid bilayer encapsulating a hydrophilic core. Using liposomes as a carrier for docetaxel improves its solubility and stability.2) Polymer carriers [Raza K, Kumar N, Misra C et al. Dextran-PLGA-loaded docetaxel micelles with enhanced cytotoxicity and better pharmacokinetic profile. Int J Biol Macromol 2016;88:206-212.; Tan LW, Ma BY, Zhao Q et al. Toxicity Evaluation and Anti-Tumor Study of Docetaxel Loaded mPEG-Polyester Micelles for Breast Cancer Therapy. J Biomed Nanotechnol 2017;13:393-408.], using microparticles or nanoparticles made from polymers such as PEG (polyethylene glycol) and PLGA (poly(lactic-co-glycolic acid)) to encapsulate docetaxel. Polymer carriers can provide better drug protection and reduce drug distribution in the body, thereby reducing its toxic side effects. However, these methods also have some shortcomings. For example, liposomes have a hydrophilic core, and only the hydrophobic environment between their phospholipid bilayers can be used to carry docetaxel, but their encapsulation capacity is low. In addition, the stability of liposomes may be poor, and fusion, rupture or drug leakage may occur easily, which may affect their distribution in the body and the release of the drug. Similarly, the amount of polymer carrier encapsulated docetaxel is also low. In addition, PEG and PLGA are not naturally present in the body, and can cause the body to produce corresponding antibodies to resist their effects. Therefore, these novel preparations, while improving the solubility and effect of docetaxel, are also faced with problems such as no targeted modification, low encapsulation rate and potential safety.

[0005] The novel nanoparticle liposomes consist of a hydrophobic core composed of neutral lipids and are encapsulated by a monomolecular phospholipid membrane. Similar in structure to naturally occurring lipid droplets and lipoproteins, they can efficiently dissolve and encapsulate hydrophobic small molecules. Furthermore, the components of liposomes are naturally present in the body, resulting in excellent biocompatibility. Furthermore, their preparation is simple and efficient. If liposomes are used to deliver docetaxel or other hydrophobic drugs, they are expected to enhance drug solubility, improve bioavailability, enhance efficacy and safety, and enable targeted delivery, greatly facilitating treatment for patients.

[0006] Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a docetaxel fat body preparation and a preparation method and application thereof, wherein the fat body preparation has good bioavailability and safety and can carry targeting molecules.

[0008] The docetaxel fat body preparation provided by the present invention comprises: a single molecule phospholipid membrane and docetaxel and neutral lipid wrapped in the single molecule phospholipid membrane.

[0009] In the present invention, the monomolecular phospholipid membrane comprises one or more of phospholipids, functional polar lipids and cationic lipids;

[0010] The phospholipid is selected from one or more of 2-bis-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPC), egg yolk lecithin, soybean lecithin, dioleoylphosphatidylethanolamine, distearoylphosphatidylcholine, egg yolk lecithin, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidic acid, sodium distearoylphosphatidylglycerol, dimyristoylphosphatidylcholine, 1-stearoyl-lysophosphatidylcholine, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000, phosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and sphingomyelin;

[0011] The functional polar lipid is selected from one or more of polyethylene glycol-modified sterols, biotin-modified sterols, amino acid-modified sterols, polypeptide-modified sterols, polysaccharide-modified sterols, nucleic acid-modified sterols, polyethylene glycol-modified phospholipids, biotin-modified phospholipids, amino acid-modified phospholipids, polypeptide-modified phospholipids, polysaccharide-modified phospholipids and nucleic acid-modified phospholipids;

[0012] The cationic lipid is selected from one or more of (2,3-dioleoyl-propyl)-trimethylammonium-chloride, (2,3-dioleoyl-propyl)-trimethylamine, 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylamine hydrochloride, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride.

[0013] In the present invention, the neutral lipid is selected from one or more of fish oil, corn oil, tricaprylin, triolein, retinol ester, wax ester, sterol ester, sterol ester, castor oil, sunflower oil, soybean oil, peanut oil, clove oil, simethicone, cinnamon oil, tea oil, liquid paraffin, star anise oil, mixed fatty acid glycerides (stearin), hydrogenated vegetable oil, refined olive oil and fat-soluble vitamins.

[0014] In order to better encapsulate docetaxel, the present invention further screens and optimizes the materials of the single-molecule phospholipid membrane and the neutral phospholipids.

[0015] In some embodiments, the material of the monomolecular phospholipid membrane is phospholipid, and the neutral lipid is at least one of fish oil, corn oil, tricaprylin, and triolein.

[0016] In some specific embodiments:

[0017] The material of the monomolecular phospholipid membrane is DOPC, and the neutral lipid is fish oil.

[0018] Or the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipid is corn oil.

[0019] Or the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipid is tricaprylin.

[0020] Or the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipid is triolein.

[0021] Or the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipids are fish oil and corn oil.

[0022] Or the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipids are fish oil and tricaprylin.

[0023] Or the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipids are fish oil and triolein.

[0024] Or the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipids are corn oil and tricaprylin.

[0025] Or the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipids are corn oil and triolein.

[0026] Or the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipids are tricaprylin and triolein.

[0027] Or the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipids are fish oil, tricaprylin and triolein.

[0028] Or the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipids are corn oil, tricaprylin and triolein.

[0029] Or the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipid is fish oil, corn oil and triolein.

[0030] Or the material of the monomolecular phospholipid membrane is DOPC, and the neutral lipids are fish oil, tricaprylin and corn oil.

[0031] Or the material of the monomolecular phospholipid membrane is phosphatidylcholine, and the neutral lipid is fish oil.

[0032] Or the material of the single-molecule phospholipid membrane is phosphatidylcholine, and the neutral lipid is corn oil.

[0033] Or the material of the monomolecular phospholipid membrane is phosphatidylcholine, and the neutral lipid is tricaprylin.

[0034] Or the material of the monomolecular phospholipid membrane is phosphatidylcholine, and the neutral lipid is triolein.

[0035] Or the material of the single-molecule phospholipid membrane is phosphatidylcholine, and the neutral lipid is fish oil and corn oil.

[0036] Or the material of the single-molecule phospholipid membrane is phosphatidylcholine, and the neutral lipid is fish oil and tricaprylin.

[0037] Or the material of the single-molecule phospholipid membrane is phosphatidylcholine, and the neutral lipid is fish oil and triolein.

[0038] Or the material of the single-molecule phospholipid membrane is phosphatidylcholine, and the neutral lipid is corn oil and tricaprylin.

[0039] Or the material of the single-molecule phospholipid membrane is phosphatidylcholine, and the neutral lipid is corn oil and triolein.

[0040] Or the material of the monomolecular phospholipid membrane is phosphatidylcholine, and the neutral lipids are tricaprylin and triolein.

[0041] Or the material of the monomolecular phospholipid membrane is phosphatidylcholine, and the neutral lipid is fish oil, tricaprylin and triolein.

[0042] Or the material of the monomolecular phospholipid membrane is phosphatidylcholine, and the neutral lipid is corn oil, tricaprylin and triolein.

[0043] Or the material of the monomolecular phospholipid membrane is phosphatidylcholine, and the neutral lipid is fish oil, corn oil and triolein.

[0044] Or the material of the single-molecule phospholipid membrane is phosphatidylcholine, and the neutral lipid is fish oil, tricaprylin and corn oil.

[0045] Or the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipid is fish oil.

[0046] Or the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipid is corn oil.

[0047] Or the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipid is tricaprylin.

[0048] Or the material of the monomolecular phospholipid membrane is phosphatidic acid, and the neutral lipid is triolein.

[0049] Or the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipid is fish oil and corn oil.

[0050] Or the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipids are fish oil and tricaprylin.

[0051] Or the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipid is fish oil and triolein.

[0052] Or the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipids are corn oil and tricaprylin.

[0053] Or the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipids are corn oil and triolein.

[0054] Or the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipids are tricaprylin and triolein.

[0055] Or the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipid is fish oil, tricaprylin and triolein.

[0056] Or the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipids are corn oil, tricaprylin and triolein.

[0057] Or the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipid is fish oil, corn oil and triolein.

[0058] Or the material of the single-molecule phospholipid membrane is phosphatidic acid, and the neutral lipids are fish oil, tricaprylin and corn oil.

[0059] Or the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipid is fish oil.

[0060] Or the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipid is corn oil.

[0061] Or the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipid is tricaprylin.

[0062] Or the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipid is triolein.

[0063] Or the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are fish oil and corn oil.

[0064] Or the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are fish oil and tricaprylin.

[0065] Or the materials of the monomolecular phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are fish oil and triolein.

[0066] Or the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are corn oil and tricaprylin.

[0067] Or the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are corn oil and triolein.

[0068] Or the materials of the monomolecular phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are tricaprylin and triolein.

[0069] Or the materials of the monomolecular phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are fish oil, tricaprylin and triolein.

[0070] Or the materials of the monomolecular phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are corn oil, tricaprylin and triolein.

[0071] Or the materials of the single-molecule phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are fish oil, corn oil and triolein.

[0072] Or the materials of the monomolecular phospholipid membrane are DOPC and phosphatidic acid, and the neutral lipids are fish oil, tricaprylin and corn oil.

[0073] In some embodiments, the volume ratio of fish oil to tricaprylin in the neutral lipid is 1:(0.1-10). In some specific embodiments, the volume ratio of fish oil to tricaprylin is 1:(0.5-5). More specifically, the volume ratio of fish oil to tricaprylin is 1:(0.8-2). For example, the volume ratio of fish oil to tricaprylin is 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2.0.

[0074] In some embodiments, the mass ratio of docetaxel, neutral lipid and phospholipid is 1:(20-200):(10:100).

[0075] In some specific embodiments, the mass ratio of docetaxel, neutral lipid and phospholipid is 1: (20-100): (10-50).

[0076] More specifically, the mass ratio of docetaxel, neutral lipid and phospholipid is 1:(20-50):(10-25).

[0077] Preferably, the mass ratio of docetaxel, neutral lipid and phospholipid is 1:(30-40):(15-20).

[0078] Preferably, the mass ratio of docetaxel, neutral lipid and phospholipid is 1:30:(15-20), or 1:31:(15-20), or 1:32:(15-20), or 1:33:(15-20), or 1:34:(15-20), or 1:35:(15-20), or 1:36:(15-20), or 1:37:(15-20), or 1:38:(15-20), or 1:39:(15-20), or 1:40:(15-20).

[0079] More preferably, the mass ratio of docetaxel, neutral lipid and phospholipid is 1:33:15, or 1:33:16, or 1:33:17, or 1:33:18, or 1:33:19, or 1:33:20, or 1:34:15, or 1:34:16, or 1:34:17, or 1:34:18, or 1:34:19, or 1:34:20, or 1:35:15, or 1:35:16, or 1:35:17, or 1:35:18, or 1:35:19, or 1:35:20.

[0080] Compared with other conditions, the neutral lipid is fish oil and tricaprylin with a volume ratio of 1:1, and the single-molecule phospholipid membrane is DOPC, which can achieve a higher encapsulation efficiency, an average particle size within 200nm, and good stability even for long-term placement.

[0081] Furthermore, the docetaxel fat body preparation of the present invention further comprises a targeting molecule, which targets and recognizes organs, tissues or cells.

[0082] In the present invention, the organs, tissues or cells are from the human body or animal body;

[0083] The organs are endocrine organs, digestive organs, circulatory organs, urinary organs, reproductive organs, locomotor organs, the nervous system, and sensory organs. Endocrine organs include the thyroid gland and pancreas. Digestive organs include the stomach, liver, gallbladder, spleen, pancreas, small intestine, and large intestine. Respiratory organs include the lungs. Circulatory organs include the heart and blood vessels. Urinary organs include the kidneys, ureters, and bladder. Reproductive organs include the uterus and ovaries. Locomotor organs include muscles and bones. The nervous system includes the cerebrum and cerebellum. Sensory organs include the skin, eyes, and ears.

[0084] The tissue or cell is derived from a tumor of the human or animal body. The tumor includes lung cancer, kidney cancer, laryngeal cancer, liver cancer, muscle tissue cancer, blood tumor, bone cancer, brain cancer, breast cancer, cervical cancer, oral or nasal mucosal cancer, bladder cancer, central nervous system cancer, cervical cancer, head and neck cancer, colon cancer, endometrial cancer, external genital cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, genitourinary tract cancer, head cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, melanoma, testicular cancer and / or thyroid cancer.

[0085] The targeting molecule can be embedded in the monomolecular phospholipid membrane, or it can be bound to the phospholipids on the monomolecular phospholipid membrane through the avidin-biotin system, or it can be connected to a substance that specifically targets phospholipids to bind to the monomolecular phospholipid membrane, or it can be a combination of any two or more of the above methods.

[0086] In the present invention, the targeting molecule is at least one of LTA-P33, ApoE, BCMA antibody, Nrp-B, Trf-B, LDLR-B, ErbB2-B, CXCR4-B, GRP78-B or Soma-B.

[0087] In some embodiments, the targeting molecule is linked to biotin, and a phospholipid molecule labeled with streptavidin is added during the preparation of the adipocytes. Alternatively, the targeting molecule is linked to streptavidin, and a phospholipid molecule labeled with biotin is added during the preparation of the adipocytes.

[0088] In some embodiments, the targeting molecule is connected to a peptide segment that targets and recognizes a single molecule of phospholipid membrane, which includes at least one of AAMB, ALDI, CYB5R3-N, LDAMP1, HSD17B13-N28, MDT-28-P, MLDS-P, DHS-3-P, HSD17B11-N28, PspA-H1, Vipp1-H1, Snf7-H1, Chmp1B-H1, PB, PE, and PF. The targeting molecule and the peptide segment that targets and recognizes a single molecule of phospholipid membrane can be connected via a linker or directly connected without a linker, which is not limited by the present invention. The linker is a cleavable linker or a self-cleaving linker. The amino acid sequence of the cleavable linker is LEAGCKNFFPRSFTSCGSLE, and the self-cleaving linker is P2A, T2A, or E2A.

[0089] In order to achieve good drug loading and at the same time make the preparation have a targeting effect, neutral lipids and phospholipids were optimized and screened.

[0090] In some embodiments, in the docetaxel fat body preparation containing the targeting molecule, the neutral lipid is selected from at least one of fish oil, tricaprylin and triolein, the phospholipid is DOPC, and the phospholipid may further include a phospholipid labeled with biotin.

[0091] The mass ratio of docetaxel, neutral lipid, phospholipid and targeting molecule is 1:(20-200):(10-100):(2-20).

[0092] In some specific embodiments, the mass ratio of docetaxel, neutral lipid, phospholipid and targeting molecule is 1:(20-100):(10-50):(2-15).

[0093] More specifically, the mass ratio of docetaxel, neutral lipid, phospholipid and targeting molecule is 1:(20-50):(10-25):(2-10).

[0094] Preferably, the mass ratio of docetaxel, neutral lipid, phospholipid and targeting molecule is 1:(30-40):(15-20):(2-5).

[0095] Preferably, the mass ratio of docetaxel, neutral lipid, phospholipid and targeting molecule is 1:30:(15-20):(2-5), or 1:31:(15-20):(2-5), or 1:32:(15-20):(2-5), or 1:33:(15-20):(2-5), or 1:34:(15-20):(2-5), or 1:35:(15-20):(2-5), or 1:36:(15-20):(2-5), or 1:37:(15-20):(2-5), or 1:38:(15-20):(2-5), or 1:39:(15-20):(2-5), or 1:40:(15-20):(2-5).

[0096] More preferably, the mass ratio of docetaxel, neutral lipid, phospholipid and targeting molecule is 1:33:15:(2-5), or 1:33:16:(2-5), or 1:33:17:(2-5), or 1:33:18:(2-5), or 1:33:19:(2-5), or 1:33:20:(2-5), or 1:34:15:(2-5), or 1:34:16:(2-5), or 1:34:17:(2~5), or 1:34:18:(2~5), or 1:34:19:(2~5), or 1:34:20:(2~5), or 1:35:15:(2~5), or 1:35:16:(2~5), or 1:35:17:(2~5), or 1:35:18:(2~5), or 1:35:19:(2~5), or 1:35:20:(2~5).

[0097] Further preferably, the mass ratio of docetaxel, neutral lipid, phospholipid and targeting molecule is 1:33:15:2, or 1:33:16:2, or 1:33:17:2, or 1:33:18:2, or 1:33:19:2, or 1:33:20:2, or 1:34:15:2, or 1:34:16:2, or 1:34:17:2, or 1:34:18:2, or 1:34:1 9:2, or 1:34:20:2, or 1:35:15:2, or 1:35:16:2, or 1:35:17:2, or 1:35:18:2, or 1:35:19:2, or 1:35:20:2, or 1:33:15:3, or 1:33:16:3, or 1:33:17:3, or 1:33:18:3, or 1:33:19:3, or 1:33:20 :3, or 1:34:15:3, or 1:34:16:3, or 1:34:17:3, or 1:34:18:3, or 1:34:19:3, or 1:34:20:3, or 1:35:15:3, or 1:35:16:3, or 1:35:17:3, or 1:35:18:3, or 1:35:19:3, or 1:35:20:3, or 1:33:15: 4, or 1:33:16:4, or 1:33:17:4, or 1:33:18:4, or 1:33:19:4, or 1:33:20:4, or 1:34:15:4, or 1:34:16:4, or 1:34:17:4, or 1:34:18:4, or 1:34:19:4, or 1:34:20:4, or 1:35:15:4, or 1:35:16:4, Or 1:35:17:4, or 1:35:18:4, or 1:35:19:4, or 1:35:20:4, or 1:33:15:5, or 1:33:16:5, or 1:33:17:5, or 1:33:18:5, or 1:33:19:5, or 1:33:20:5, or 1:34:15:5, Or 1:34:16:5, or 1:34:17:5, or 1:34:18:5, or 1:34:19:5, or 1:34:20:5, or 1:35:15:5, or 1:35:16:5, or 1:35:17:5, or 1:35:18:5, or 1:35:19:5, or 1:35:20:5.

[0098] Compared with other phospholipids and neutral lipids, the solution in the embodiment of the present invention is more conducive to improving the encapsulation efficiency and stability of the preparation. It can also ensure that the efficacy of the preparation is not affected and the targeting is better.

[0099] In some specific embodiments,

[0100] The targeting molecule is LTA-P33;

[0101] The neutral lipids are fish oil and tricaprylin;

[0102] The monomolecular phospholipid membrane is 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine;

[0103] The volume ratio of the fish oil to tricaprylin is 1:(0.1-10). Preferably, the volume ratio of the fish oil to tricaprylin is 1:1.

[0104] In some specific embodiments, the targeting molecule is ApoE;

[0105] The neutral lipids are fish oil and tricaprylin;

[0106] The monomolecular phospholipid membrane is 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine;

[0107] The volume ratio of the fish oil to tricaprylin is 1:(0.1-10). Preferably, the volume ratio of the fish oil to tricaprylin is 1:1.

[0108] In some specific embodiments, the targeting molecule is a BCMA antibody, and the BCMA is labeled with streptavidin;

[0109] The neutral lipid is triolein;

[0110] The monomolecular phospholipid membrane is a phospholipid labeled with 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine and biotin, wherein the phospholipid labeled with biotin is one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin, and sphingomyelin. In some embodiments, the biotin-labeled phospholipid is biotin-labeled phosphatidylcholine (18:1 Biotinyl Cap PE, referred to as Bio-PE).

[0111] In the monomolecular phospholipid membrane, the mass ratio of the 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPC) to the biotin-labeled phospholipid is (0-200):(0.05-200).

[0112] In some embodiments, the mass ratio of DOPC to biotin-labeled phospholipid is (100-200):(0.05-10) or (150-200):(0.05-0.1). In some specific embodiments, the mass ratio of DOPC to biotin-labeled phospholipid is (100-150):(0.05-1). Preferably, the mass ratio of DOPC to biotin-labeled phospholipid is 145:0.08.

[0113] Furthermore, the present invention provides a method for preparing the aforementioned docetaxel fat body preparation without the targeting molecule, comprising:

[0114] Step 1: mixing a docetaxel solution and a neutral lipid, and removing the solvent to obtain a neutral lipid containing docetaxel;

[0115] Step 2: The neutral lipids and phospholipids containing docetaxel are mixed, and the docetaxel-loaded fat bodies are obtained after repeated vortexing and centrifugation.

[0116] Furthermore, the present invention provides a method for preparing the docetaxel fat body preparation containing the targeting molecule as described above, comprising:

[0117] Step A: mixing a docetaxel solution and a neutral lipid, and removing the solvent to obtain a neutral lipid containing docetaxel;

[0118] Step B: mixing the targeting molecule solution with the phospholipid, and removing the solvent to obtain the phospholipid containing the targeting molecule;

[0119] Step C: The neutral lipid containing docetaxel and the phospholipid containing the targeting molecule are mixed, and the docetaxel-encapsulated fat body is obtained after repeated vortexing and centrifugation.

[0120] Or further, the present invention provides a method for preparing the docetaxel fat body preparation containing the targeting molecule as described above, comprising:

[0121] Step a: mixing a docetaxel solution and a neutral lipid, and removing the solvent to obtain a neutral lipid containing docetaxel;

[0122] Step b: mixing the neutral lipid containing docetaxel with a buffer and phospholipids, and repeatedly vortexing and centrifuging;

[0123] Step c: mixing with targeting molecules and incubating to obtain docetaxel-loaded fat bodies.

[0124] As described above, in the preparation method,

[0125] The solvent in the docetaxel solution is an organic solvent. The organic solvent is any one of anhydrous ethanol, chloroform, methanol, benzene, toluene, xylene, butanol, isopropanol, ethyl ether, acetone, cyclohexanone, methyl isobutyl ketone, ethyl acetate, butyl acetate, cyclohexanone, or petroleum ether, or a combination of two or more thereof; preferably, anhydrous ethanol.

[0126] The solvent in the targeting molecule solution is an organic solvent. The organic solvent is a mixture of methanol and at least one of the following solvents: the solvent includes anhydrous ethanol, chloroform, benzene, toluene, xylene, butanol, isopropanol, ether, acetone, cyclohexanone, methyl isobutyl ketone, ethyl acetate, butyl acetate, cyclohexanone or petroleum ether, preferably a mixture of methanol and chloroform;

[0127] The buffer solution is PBS buffer solution, HEPES buffer solution, sucrose solution, NaCl solution, KCl solution, MgCl2 solution, etc.

[0128] In the preparation method as described above, the repeated vortexing and centrifugation include:

[0129] After mixing, the mixture was vortexed to obtain Mixture 1.

[0130] In this step, the parameters of the vortex include: vortexing at 3000-4000 rpm for 3-7 minutes, operating for 1-10 seconds, and resting for 1-10 seconds. Preferably, the vortex speed is 3000, 3200, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 rpm. Preferably, the vortex duration is 3 minutes, 4 minutes, 5 minutes, 6 minutes, or 7 minutes. Preferably, the vortex stops for 1 second every working 1 second, or stops for 2 seconds every working 2 seconds, or stops for 2 seconds every working 2 seconds, or stops for 3 seconds every working 3 seconds, or stops for 4 seconds every working 4 seconds, or stops for 5 seconds every working 5 seconds, or stops for 6 seconds every working 6 seconds, or stops for 7 seconds every working 7 seconds, or stops for 8 seconds every working 8 seconds, or stops for 9 seconds every working 9 seconds, or stops for 10 seconds every working 10 seconds, or stops for 6 seconds every working 5 seconds, or stops for 7 seconds every working 5 seconds, or stops for 8 seconds every working 5 seconds, or stops for 9 seconds every working 5 seconds, or stops for 10 seconds every working 6 seconds, or stops for 7 seconds every working 6 seconds, or stops for 8 seconds every working 6 seconds, or stops for 9 seconds every working 6 seconds, or stops for 10 seconds every working 6 seconds, or stops for 8 seconds every working 7 seconds, or stops for 9 seconds every working 7 seconds, or stops for 10 seconds every working 8 seconds, or stops for 10 seconds every working 8 seconds, or stops for 10 seconds every working 9 seconds. In some specific embodiments, the vortex parameters in this step include 4000 rpm, vortexing for 10 seconds, stopping for 10 seconds, and vortexing for 4 minutes. Alternatively, the vortex parameters in this step include 4000 rpm, vortexing for 10 seconds, stopping for 5 seconds, and vortexing for 3 minutes.

[0131] The mixture 1 was centrifuged to collect the lower layer solution, and then vortexed again to obtain a mixture 2.

[0132] In this step, the vortexing parameters include 1000-4000 rpm, and the centrifugation parameters include: centrifugation at 800-1200 g for 3-7 min at room temperature. Preferably, the centrifugation speed is 800 g, 900 g, 1000 g, 1100 g, or 1200 g, and the centrifugation time is 3 min, 4 min, 5 min, 6 min, or 7 min. In some embodiments, the centrifugation conditions include centrifugation at 1000 g for 5 min at room temperature.

[0133] After the mixture 2 is centrifuged to remove the precipitate, it is vortexed again to obtain the mixture 3;

[0134] In this step, the vortexing parameters include 1000-4000 rpm, and the centrifugation parameters include: in actual application, centrifugation at 18000-22000g for 3-7 minutes. Preferably, the centrifugation speed is 18000g, 19000g, 20000g, 21000g or 22000g, and the centrifugation time is 3 minutes, 4 minutes, 5 minutes, 6 minutes or 7 minutes. In some embodiments, the centrifugation conditions include centrifugation at 20000g for 5 minutes at room temperature.

[0135] The mixture 3 is centrifuged to collect the lower layer solution, and vortexed again to obtain the mixture 4 containing the fat body. In this step, the vortex parameters include 1000-4000 rpm, and the centrifugation parameters include: in actual application, centrifugation at 800-1200g for 3-7 minutes. Preferably, the centrifugation speed is 800g, 900g, 1000g, 1100g, or 1200g, and the centrifugation time is 3 minutes, 4 minutes, 5 minutes, 6 minutes, or 7 minutes. In some embodiments, the centrifugation conditions include centrifugation at 1000g for 5 minutes at room temperature.

[0136] The preparation method provided by the present invention is simple and easy to operate, and the resulting preparation has good encapsulation efficiency and stability. It has been verified that the non-targeted docetaxel fat body can inhibit the growth of various cancer cells, such as hematological tumor cells, breast cancer cells, and liver cancer cells, with an inhibitory effect superior to the clinically used albumin paclitaxel and a safety superior to the clinically used docetaxel drug (docetaxel injection). Targeted docetaxel fat body inhibits the growth of lung cancer with greater effectiveness than the clinically used docetaxel drug (docetaxel injection).

[0137] Furthermore, the above-mentioned preparation or the preparation obtained by the above-mentioned method is used in the preparation of drugs or vaccines for preventing and treating tumors.

[0138] In the present invention, the tumor includes: lung cancer, kidney cancer, laryngeal cancer, liver cancer, muscle tissue cancer, blood tumor, bone cancer, brain cancer, breast cancer, cervical cancer, oral or nasal mucosal cancer, bladder cancer, central nervous system cancer, cervical cancer, head and neck cancer, colon cancer, endometrial cancer, external genital cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, genitourinary tract cancer, head cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, melanoma, testicular cancer and / or thyroid cancer.

[0139] Furthermore, the present invention provides a medicine or vaccine, which includes the preparation as described above, or the preparation prepared by the method as described above.

[0140] The medicine or vaccine of the present invention also includes pharmaceutically acceptable excipients.

[0141] The dosage form of the medicine or vaccine of the present invention is oral preparation, inhalant or injection.

[0142] Optionally, the drug or vaccine is in the form of an oral preparation, for example, a tablet, a pill, an oral solution, a capsule, a syrup, a dropper or a granule.

[0143] In some embodiments provided herein, the capsule is a hard capsule or a soft capsule.

[0144] In some embodiments provided herein, the tablet is an oral tablet or buccal tablet.

[0145] Oral tablets are tablets for oral administration. Most of the drugs in these tablets are absorbed through the gastrointestinal tract to exert their effects, while some drugs in other tablets exert their effects locally in the gastrointestinal tract. In some embodiments provided herein, the oral tablets are conventional compressed tablets, dispersible tablets, effervescent tablets, chewable tablets, coated tablets, or sustained-release tablets.

[0146] The medicine or vaccine is inhaled, and optionally, it is an inhalation aerosol, an inhalation powder, or a liquid preparation for use in a nebulizer.

[0147] The medicine or vaccine is an injection, for example, an injection solution or an injection powder.

[0148] The medicine or vaccine of the present invention further comprises an effective amount of a tumor inhibitor;

[0149] The tumor suppressors include: cisplatin, carboplatin, oxaliplatin, 5-fluorouracil (5-FU), methotrexate, daunorubicin, dactinomycin-D, irinotecan (CPT-11), mitoxantrone, estramustine, vincristine, dexamethasone, prednisone, lomustine, methotrexate, pirarubicin, doxorubicin, gemcitabine, quizartinib or bevacizumab.

[0150] The present invention also provides a method for preventing and treating tumors, comprising administering the aforementioned drug or vaccine, wherein the administration method includes oral administration, inhalation and / or injection.

[0151] The subject of the method is a human or, the subject of the method is a primate or a non-primate mammal.

[0152] The present invention provides novel nanoparticle fat bodies (having a hydrophobic core) that encapsulate docetaxel to construct docetaxel fat bodies, which exhibit excellent anti-tumor effects and are safer than the clinically used docetaxel drug (docetaxel injection). Furthermore, the docetaxel fat bodies undergo targeted surface modification, effectively inhibiting the growth of lung cancer, liver cancer, and hematologic tumors, with significantly better inhibitory effects than the clinically used docetaxel drug (docetaxel injection), and also possessing superior safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0153] Figure 1 shows that fat bodies have good biocompatibility. Serum biochemical indicators, body weight changes, and liver tissue sections of mice treated with saline or fat bodies for 50 days: A, body weight change; B, alanine aminotransferase (ALT); C, aspartate aminotransferase (AST); D, creatinine (Cre); E, urea (Urea); F, hemoglobin (HGB); G, red blood cell count (RBC); H, low-density lipoprotein cholesterol (LDL-C); I, triglycerides (TAG). J, liver tissue section, Bar = 200 μm. ** indicates p < 0.01, *** indicates p < 0.001.

[0154] Figure 2: Preparation of non-targeted docetaxel fat bodies, including: A, preparation process of docetaxel fat bodies; B, HPLC detection of DTX free standard in ethanol and DTX in docetaxel fat bodies; C, TLC detection of DTX signal in docetaxel fat bodies, the dotted box indicates the location of DTX standard; D, optical microscopy observation of the morphology of blank fat bodies and docetaxel fat bodies, scale bar is 5 μm;

[0155] Figure 3 shows that docetaxel fat bodies have good structural stability, where: A, dynamic light scattering measurement of the average particle size change of docetaxel fat bodies with different contents after storage for different time periods; B, dynamic light scattering measurement of the PDI (polydispersity index) change of docetaxel fat bodies with different contents after storage for different time periods; C, change of drug loading (DL) of docetaxel fat bodies with different contents after storage for different time periods; D, change of encapsulation efficiency (EE) of docetaxel fat bodies with different contents after storage for different time periods; E, change of the number of DTX molecules per adiposome (M / A) of docetaxel fat bodies with different contents after storage for different time periods; F, optical microscopy observation of the morphology and structure of docetaxel fat bodies with low DTX content, scale bar is 2 μm; G, optical microscopy observation of the morphology and structure of docetaxel fat bodies with high DTX content, scale bar is 2 μm. DTX-Ad1 is a docetaxel fat body with low DTX content, and DTX-Ad2 is a docetaxel fat body with high DTX content;

[0156] Figure 4 shows that docetaxel adipsomes have a stable structure and slow release rate. A, DTX retention of docetaxel adipsomes and docetaxel injection after different dialysis times; B, average particle size and PDI of docetaxel adipsomes after different dialysis times; C, optical micrographs of docetaxel adipsomes after different dialysis times, scale bar is 5 μm; ** indicates p < 0.01, *** indicates p < 0.001. DTX-Ad is docetaxel adipsomes, and Commercial DTX is the commercialized docetaxel drug docetaxel injection.

[0157] Figure 5: The clearance rate of docetaxel adipocytes in vivo is lower than that of docetaxel injection, including: A, photographs of centrifuged blood collected from mice after different time periods of docetaxel adipocytes and docetaxel injection in vivo. The white portion indicated by the red arrow is the docetaxel adipocytes; B, plasma was separated from mice after different time periods of docetaxel adipocytes and docetaxel injection in vivo, and the DTX content was detected; where ** indicates p < 0.01, *** indicates p < 0.001; DTX-Ad is docetaxel adipocytes, and Commercial DTX is the commercialized docetaxel drug docetaxel injection;

[0158] Figure 6 shows that docetaxel fat bodies have anti-tumor activity, wherein: docetaxel fat bodies and ethanol (control group) were treated with the corresponding cells at the indicated concentrations, and the cell survival rate was measured by CCK8; A, breast cancer cell 4T1; B, human prostate cancer cell PC3; C, human hematologic malignancy cell H929; D, human hematologic malignancy cell Raji; *** indicates p < 0.001;

[0159] Figure 7 shows that docetaxel adipocytes are more effective in killing cancer cells than the clinical drug albumin-paclitaxel. The prepared docetaxel adipocytes and the clinical drug albumin-paclitaxel were used to treat hematological tumor cells H929 (A), liver cancer cells Hepa1-6 (B), breast cancer cells 4T1 (C), and colorectal cancer cells CT26 (D) at the concentrations shown; the cell survival rates were measured using CCK8 assay; ** indicates p < 0.01, and *** indicates p < 0.001.

[0160] Figure 8 shows that docetaxel fat bodies inhibit the growth of breast cancer in mice, with a safety profile superior to the clinical drug docetaxel injection. A, changes in tumor volume in mice treated with saline, docetaxel injection, and docetaxel fat bodies; B, body weight change in mice treated with saline, docetaxel injection, and docetaxel fat bodies; C, hemolysis rate of erythrocytes treated with docetaxel injection and docetaxel fat bodies; *** indicates p < 0.001, **** indicates p < 0.0001.

[0161] Figure 9 shows that lung-targeted docetaxel adipocytes are more effective in inhibiting lung cancer than docetaxel injection, wherein: A, tissue distribution of fluorescently labeled lung-targeted docetaxel adipocytes (Lu-DTX-Ad); B, changes in lung tumor volume and tumor inhibition rate in mice after normal saline, docetaxel injection, and lung-targeted docetaxel adipocytes; *** indicates p < 0.001;

[0162] Figure 10 shows the preparation of liver-targeted docetaxel fat bodies, including: A, staining results of purified recombinant ApoE expressed in a prokaryotic system; B, optical microscopy results of liver-targeted docetaxel fat bodies, scale bar is 5 μm; C, expression of LDLR in mouse liver, hepatoma cell lines HepG2 and Hepa1-6, and embryonic kidney epithelial cells HEK293; D, tissue distribution of fluorescently labeled liver-targeted docetaxel fat bodies after injection into mice at different times;

[0163] Figure 11 shows that the effect of liver-targeted docetaxel fat body in inhibiting liver cancer is better than that of docetaxel injection, wherein: A, the size, weight and inhibition rate of liver tumor volume after treatment of liver cancer mice with normal saline group, docetaxel injection group, docetaxel fat body group and liver-targeted docetaxel fat body group; B, the body weight change rate after treatment of liver cancer mice with normal saline group, docetaxel injection group, docetaxel fat body group and liver-targeted docetaxel fat body group; C, the biochemical indicators of liver function and renal function after treatment of liver cancer mice with normal saline group, docetaxel injection group, docetaxel fat body group and liver-targeted docetaxel fat body group; wherein, * indicates p < 0.05, ** indicates p < 0.01;

[0164] Figure 12 shows the preparation and targeting of hematologic tumor cell-targeted adipomes, wherein: A, preparation of hematologic tumor cell-targeted adipomes; B, expression of BCMA in hematologic tumor cells Raji, hematologic tumor cells H929, and human embryonic kidney epithelial cells HEK293; C, flow cytometry results of fluorescently labeled non-targeted adipomes, hematologic tumor cell-targeted adipomes, and inhibition of endocytosis of targeted adipomes by hematologic tumor cells H929; D, optical microscopy results of fluorescently labeled non-targeted adipomes, hematologic tumor cell-targeted adipomes, and inhibition of endocytosis of targeted adipomes by hematologic tumor cells H929. Scale bar is 5 μm.

[0165] Figure 13 shows that hematologic malignancy cell-targeted docetaxel adipomes are more effective than docetaxel injection: H929 cells were treated with the same concentration of 20 ng / ml of DTX, including untargeted docetaxel adipomes, hematologic malignancy cell-targeted docetaxel adipomes, and the clinical drug docetaxel injection, for 2, 4, and 16 hours. The culture medium was removed and replaced with drug-free culture medium. Cell survival was measured using CCK8 assay. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. DETAILED DESCRIPTION

[0166] The present invention provides a docetaxel fat body formulation and its preparation method and application. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0167] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0168] In this application, the term "and / or" describes the association relationship between associated objects, indicating that three relationships may exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0169] The terms "include," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0170] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0171] The term "drug" herein refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective and that contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition is administered.

[0172] The term "prevent and treat" herein includes prevention and / or treatment. The "treatment" refers to surgical or pharmaceutical treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as cancer and tumors. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, weakening of the disease extent, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Objects in need of treatment include objects already suffering from a condition or disease, as well as objects susceptible to a condition or disease, or objects intended to prevent a condition or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0173] The term "administered" herein refers to an organism that receives treatment for a particular disease or condition as described herein. Exemplarily, the subject receiving treatment for a disease or condition is a mammal, such as a human, a primate (eg, monkey), or a non-primate mammal.

[0174] The term "subject" herein refers to an organism that is being treated for a particular disease or condition as described herein. Exemplarily, a "subject" includes a mammal, such as a human, primate (e.g., monkey), or non-primate mammal, being treated for a disease or condition.

[0175] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0176] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the terms "tumor" or "neoplasm" refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0177] The term "IC50" in this article refers to the half-inhibitory concentration of the antagonist being measured. It can be understood that a certain concentration of a drug induces 50% tumor cell death. This concentration is called the 50% inhibitory concentration, that is, the concentration corresponding to the ratio of dead cells to total cells is equal to 50%. The IC50 value can be used to measure the ability of a drug to induce death. That is, the stronger the induction ability, the lower the value.

[0178] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0179] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0180] The fragment names and sequences involved in this article are as follows:

[0181] Table 1 Names and amino acid sequences of peptides targeting single-molecule phospholipid membranes

[0182] Table 2 Targeting molecule names and amino acid sequences and targeting proteins

[0183] The present invention uses a novel nano-carrier fat body with a hydrophobic core structure to encapsulate the docetaxel drug, which has a 100-fold increase in solubility in water and has biological activity, significantly killing multiple cancer cells, with better effectiveness than the clinical drug albumin paclitaxel and better safety than the clinical drug docetaxel injection. In addition, the present invention uses different methods to target the surface of the docetaxel fat body, including lung targeting, liver targeting, and blood cancer cell targeting, effectively inhibiting the growth of lung cancer, liver cancer, and blood tumors. The inhibitory effect is significantly better than the clinically used docetaxel drug (docetaxel injection), and its safety is also better than docetaxel injection. The present invention is further described below in conjunction with the examples.

[0184] Example 1 Fat body has good biocompatibility

[0185] The biocompatibility of nanoparticles is the cornerstone of clinical translation. To demonstrate that the novel nanoparticle-based fat bodies are a safe carrier for delivering hydrophobic small molecule drugs, we tested the biocompatibility of empty fat bodies (fat bodies without any drug). The preparation method of empty fat bodies is as follows:

[0186] 1) Neutral lipids: Mix fish oil and tricaprylin (8:0 TAG) in a volume ratio of 1 / 1.

[0187] 2) 80 μl of 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine solution (containing 2 mg DOPC) was added to a microcentrifuge tube, and the solvent was blown dry with high-purity nitrogen gas.

[0188] 3) Add 100 μl of PBS and 5 μl of the neutral lipid from step 1) to a microcentrifuge tube and vortex for 4 minutes (vortex for 10 seconds, rest for 10 seconds) (in actual application, vortex for 3-7 minutes, vortexing conditions are 3000-4000 rpm, and 4000 rpm is used in this example) to obtain a milky white lipid mixture 1. The lipid mixture 1 is centrifuged at 1000 g for 5 minutes (in actual application, centrifugation at 800-1200 g for 3-7 minutes is acceptable). After centrifugation, the liquid phase system presents two layers. The lower milky white solution is collected by extraction and vortexed to obtain a milky white lipid mixture 2.

[0189] 4) The lipid mixture 2 obtained in step 3) was centrifuged at 20,000 g for 5 min (in practice, 18,000-22,000 g for 3-7 min is acceptable). After centrifugation, the precipitate at the bottom of the microcentrifuge tube was removed and vortexed to obtain a milky white lipid mixture 3.

[0190] 5) The lipid mixture 3 obtained in step 4) is centrifuged at 1000 g for 5 minutes (in actual application, 800-1200 g for 3-7 minutes is acceptable). After centrifugation, the liquid phase system presents two layers. The lower milky white solution is collected by extraction and vortexed to obtain a milky white lipid mixture 4, which is the final fat body.

[0191] We injected the prepared empty fat bodies and saline into mice via the tail vein twice a week, 200 μl each time (OD600 = 1.4), for 50 days. There was no difference in body weight between the two groups, nor in serum biochemical parameters or blood count (Figures 1A-G), including liver inflammation markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST), kidney inflammation markers creatinine (Cre) and urea (Urea), and blood count markers hemoglobin (HGB) and red blood cell count (RBC). Furthermore, compared with the saline-treated group, serum levels of "bad" LDL-C in mice treated with fat bodies were significantly decreased (Figure 1H), while TAG levels remained unchanged (Figure 1I), suggesting that fat bodies have the potential to improve cardiovascular disease. Liver tissue sections from both groups showed normal morphology of the central vein and hepatocytes (Figure 1J). These results demonstrate the excellent biocompatibility of fat bodies, laying the foundation for their clinical translation.

[0192] Example 2 Preparation, stability, biological activity and safety of non-targeted docetaxel fat bodies

[0193] 1. Preparation of non-targeted docetaxel adiposome (DTX-adiposome, DTX-Ad)

[0194] The construction method is as follows:

[0195] 1) Neutral lipids: Mix fish oil and tricaprylin (8:0 TAG) in a volume ratio of 1 / 1.

[0196] 2) Dissolve docetaxel (DTX) in anhydrous ethanol to a concentration of 20 mg / ml.

[0197] 3) 200 μl of the neutral lipid prepared in different volume ratios was taken and added to 200 μl of docetaxel solution, and the ethanol was blown dry with high-purity nitrogen to obtain neutral lipid containing DTX.

[0198] 4) 80 μl of 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine solution (containing 2 mg DOPC) was added to a microcentrifuge tube, and the solvent was blown dry with high-purity nitrogen gas.

[0199] 5) Add 100 μl of PBS and 5 μl of the neutral lipid containing DTX prepared in step 3) to a microcentrifuge tube and vortex for 4 minutes (vortex for 10 seconds, rest for 10 seconds) (in actual application, vortex for 3-7 minutes, vortexing conditions are 3000-4000 rpm, and 4000 rpm is used in this example) to obtain a milky white lipid mixture 1. The lipid mixture 1 is centrifuged at 1000 g for 5 minutes (in actual application, centrifugation at 800-1200 g for 3-7 minutes is acceptable). After centrifugation, the liquid phase system exhibits two stratifications. The lower milky white solution is collected by extraction and vortexed to obtain a milky white lipid mixture 2.

[0200] 6) The lipid mixture 2 obtained in step 5) was centrifuged at 20,000 g for 5 minutes (in actual application, 18,000-22,000 g for 3-7 minutes is acceptable). After centrifugation, the precipitate at the bottom of the microcentrifuge tube was removed and vortexed to obtain a milky white lipid mixture 3.

[0201] 7) The lipid mixture 3 obtained in step 6) was centrifuged at 1000 g for 5 minutes (in actual application, 800-1200 g for 3-7 minutes is acceptable). After centrifugation, the liquid phase system showed two layers. The lower milky white solution was collected by extraction and vortexed to obtain a milky white lipid mixture 4, which was the final fat body carrying the hydrophobic small molecule compound docetaxel (docetaxel fat body).

[0202] The presence of docetaxel in the docetaxel liposomes was determined by HPLC (high-performance liquid chromatography) and TLC (thin-layer chromatography), respectively. HPLC detection conditions were: a 10 μl sample volume, a mobile phase of methanol:water = 7:3 (volume ratio), a flow rate of 1.0 ml / min, and a UV detection wavelength of 254 nm. TLC detection was performed by adding the prepared docetaxel liposomes to an equal volume of methanol and two volumes of chloroform to extract lipids. The organic phase was collected and dried with nitrogen to obtain total lipids. The total lipids were added to 100 μl of chloroform, and 10 μl was loaded onto a silica gel plate. The plate was developed in a solvent consisting of n-hexane:diethyl ether:glacial acetic acid (volume ratio 80:20:1) to separate TAGs. The plate was then developed in a solvent consisting of chloroform:methanol:glacial acetic acid:water (volume ratio 75:13:9:3) to separate DOPC and DTX.

[0203] The elution time of DTX in the docetaxel fat body is consistent with the elution time of free DTX standard substances in ethanol, all is 7.5min (B among Fig. 2). According to the position (shown in the dotted line frame) of DTX standard substances, judge whether there is the signal of DTX in the fat body in the TLC detection result, in the swimming lane of DTX fat body 1 and DTX fat body 2, there is signal at the position same as the DTX standard substances, and in blank fat body, there is not the signal of DTX (C among Fig. 2). It is 110nm that dynamic light scattering instrument has detected the average particle size of blank fat body, and PDI (polydispersity coefficient) is 0.15. The average particle size of docetaxel fat body is 118nm and PDI is 0.11. In addition, by optical microscope observation blank fat body and the morphological structure of docetaxel fat body, both are homogeneous spherical structures, and do not have the pollution (D among Fig. 2) of other membrane impurities. The above results show, successfully prepared the fat body that contains docetaxel, and its purity is high, and homogeneity is good.

[0204] 2. Docetaxel fat bodies have good stability

[0205] To test the stability and encapsulation efficiency of docetaxel adiposomes, we prepared four tubes of low-DTX-content docetaxel adiposomes (DTX-Ad1) and four tubes of high-DTX-content docetaxel adiposomes (DTX-Ad2). These tubes were stored at 4°C for 42 days and tested regularly to assess the properties of the docetaxel adiposomes. Particle size, polydispersity index (PDI), encapsulation efficiency (EE), drug loading (DL), and the number of DTX molecules per adiposome (M / A) were also measured weekly to assess the stability of the samples. During 42 days of monitoring, the particle size of docetaxel adipsomes containing both low and high DTX contents remained stable, ranging from 100 to 150 nm (Figure 3A), and the PDI remained consistently below 0.2 (Figure 3B). Encapsulation efficiency (EE), drug loading (DL), and the number of DTX molecules per adiposome (Molecules / Adiposome, M / A) were calculated using the following formulas: DTX-Ad1 had a drug loading of approximately 0.3% and an encapsulation efficiency of approximately 30%. DTX-Ad2 had a drug loading of approximately 1.4% and an encapsulation efficiency of approximately 40%. Furthermore, the drug loading and encapsulation efficiency remained relatively stable after different storage times (Figures 3C and 3D). Similarly, after the DTX-Ad1 and DTX-Ad2 samples were stored for different periods of time, the number of DTX molecules in each fat body was relatively stable, with 1500 DTX molecules per fat body and 6000 DTX molecules per fat body, respectively (Figure 3E).

[0206] Furthermore, optical microscopic observation of the morphological structure of the docetaxel fat bodies after 1 day and 42 days of storage revealed uniform spherical structures without contamination by other membrane impurities (Figure 3F and G), indicating that the docetaxel fat bodies were of high purity, good uniformity, and good structural stability.

[0207] We also further measured the stability and release rate of docetaxel adipsomes under room-temperature shaking conditions. We placed docetaxel adipsomes (DTX-Ad) and commercial docetaxel injection (Commercial DTX) in dialysis cups and shook them at 200 rpm at room temperature. Samples were periodically taken from the cups to measure DTX content and to photograph the particle size and morphology of the docetaxel adipsomes. The adipsomes were then filled to the initial volume with the same PBS. The results showed that docetaxel adipsomes were stable and that DTX encapsulation reduced the release rate. After 14 days of dialysis, the DTX content in the adipsomes remained at approximately 68%, while no DTX was detected in the commercial docetaxel injection (Figure 4A). The DTX-Ad particle size remained stable, consistently within 200 nm, and the PDI was within 0.2 (Figure 4B). Optical microscopy of samples taken at different time points also showed that large and small docetaxel fat bodies were evenly distributed and had intact morphology (Figure 4C). These results demonstrate that under room temperature oscillation conditions, docetaxel fat bodies are stable and release at a significantly slower rate than docetaxel injection, achieving a sustained-release effect.

[0208] 3. The clearance rate of docetaxel fat body in the body is lower than that of docetaxel injection

[0209] Further, we detect the metabolic rate of docetaxel fat body in vivo. The tail vein of mice was injected with equal amounts of docetaxel injection and docetaxel fat body respectively, at a concentration of 15 mg / kg, with 4 mice in each group, a total of 24 mice. After 10, 30 and 60 minutes after injection respectively, mice were killed, the plasma of mice was separated, and the content change of DTX was detected. As shown in Figure 5, after injection at different times, docetaxel fat body structure (A in Figure 5, the white fat body-like structure shown by the red arrow) can still be observed in the mouse serum. In addition, after injection for 60 minutes, the concentration of docetaxel in the mouse plasma processed by docetaxel fat body was significantly higher than that of docetaxel injection (B in Figure 5). The above results show that docetaxel fat body is stable in blood, and its clearance rate in vivo is lower than that of docetaxel injection, significantly prolonging the time when the drug exists in the body.

[0210] 4. Docetaxel fat bodies have anti-tumor activity

[0211] We further tested the biological activity of docetaxel fat bodies and their ability to kill tumor cells. Non-targeted docetaxel fat bodies prepared as in the previous examples were used to treat breast cancer cells 4T1 (Figure 6A), human prostate cancer cells PC3 (Figure 6B), human hematologic malignancy cells H929 (Figure 6C), and human hematologic malignancy cells Raji (Figure 6D). The results showed that compared to the untreated group, the killing effect of docetaxel fat bodies on cancer cells became increasingly better with increasing docetaxel concentration, indicating that docetaxel fat bodies have anti-tumor activity.

[0212] Cell viability assay: 3000 cells / well of the cells to be treated were plated in a 96-well plate. After overnight attachment, the cells were treated with the indicated drug concentrations and incubated for 72 hours. Subsequently, the original culture medium was replaced with culture medium containing 10% CCK8. After 1 hour of incubation, the absorbance at 450 nm was read using a microplate reader. Cell viability was calculated using the following formula:

[0213] Cell Viability=(Ae-Ab) / (Ac-Ab)*100%

[0214] Wherein, Ae represents the absorbance value of the well containing cells after drug treatment.

[0215] Ac represents the absorbance value of the wells containing cells without drug treatment.

[0216] Ab represents the absorbance value of blank wells containing only culture medium and CCK8 reagent, which is used to correct background noise.

[0217] 5. Docetaxel fat bodies are more effective in killing cancer cells than the clinical drug albumin paclitaxel

[0218] To compare the cancer cell killing effects of docetaxel fat bodies and the clinical drug albumin-paclitaxel, the prepared docetaxel fat bodies and the clinical drug albumin-paclitaxel were used to treat hematological tumor cells H929 (Figure 7A), liver cancer cells Hepa1-6 (Figure 7B), breast cancer cells 4T1 (Figure 7C), and colorectal cancer cells CT26 (Figure 7D) at the indicated concentrations. At the same drug concentration, docetaxel fat bodies were significantly more effective than albumin-paclitaxel in killing various cancer cells. Furthermore, the IC50 of docetaxel fat bodies was significantly lower than that of albumin-paclitaxel (Table 1). These results show that docetaxel fat bodies are significantly more effective than the clinical drug albumin-paclitaxel in killing various cancer cells.

[0219] Table 1: IC50 of docetaxel fat body and albumin paclitaxel in killing various cancer cells

[0220] 6. Docetaxel fat bodies inhibit the growth of breast cancer in mice and are safer than the clinical drug docetaxel injection

[0221] To highlight the advantages of the novel formulation docetaxel fat body, we further compared the efficacy and safety of docetaxel fat body with the clinical drug docetaxel injection in mice.

[0222] First, mouse breast cancer cells 4T1 were injected into the abdomen of BALB / c female mice for subcutaneous tumor bearing. The volume of the subcutaneous tumor was measured 7 days later. The maximum diameter (denoted as a, unit: mm) and the minimum diameter (denoted as b, unit: mm) of the tumor were measured with a vernier caliper. The tumor volume was calculated as (denoted as V, unit: mm) 3 ):V=a×b 2 / 2. When the volume of the subcutaneous tumor is 100mm 3 The mice were randomly divided into three groups: normal saline group (n=8), docetaxel injection group (n=10) and docetaxel fat body group (n=9). The dosage was equivalent to 15 mg / kg of docetaxel, which was intravenously administered once a week for a total of three times. The volume of the mouse tumor and the weight change were detected during the administration period. As shown in the figure, compared with the normal saline group, docetaxel fat body significantly inhibited the growth of subcutaneous tumors, and its effect was comparable to that of the clinical drug docetaxel injection (Figure 8A). However, the weight change rate results showed that the reduction in mouse body weight by docetaxel fat body was significantly less than that by docetaxel injection. The weight change rate of mice in the docetaxel injection treatment group was -16.64±2.77%, while the weight change rate of mice in the docetaxel fat body treatment group was -9.58±8.34% (Figure 8B). At the same time, erythrocyte hemolysis results showed that at different docetaxel concentrations, docetaxel injection significantly destroyed erythrocytes, with the erythrocyte hemolysis rate significantly higher than that of docetaxel fat bodies (Figure 8, C). The values ​​are shown in Table 2. Therefore, these results indicate that docetaxel fat bodies inhibit the growth of breast cancer in mice and are safer than the clinical drug docetaxel injection.

[0223] Red blood cell hemolysis test operation method:

[0224] 1) After anesthetizing a healthy mouse, approximately 1 ml of blood was collected from the eyeball and placed in an anticoagulant tube. The supernatant was removed after centrifugation at 3000 g for 5 min. The remaining 500 μl of red blood cells was added to 15 ml of PBS and mixed by pipetting to obtain a red blood cell suspension.

[0225] 2) Take 500 μl of the red blood cell suspension obtained in step (1) and add it to 500 μl of pure water as a positive control, and add it to 500 μl of PBS as a negative control, and mix them.

[0226] 3) Dilute the DTX concentration in docetaxel injection and docetaxel fat body to 12 μg / ml, 30 μg / ml and 60 μg / ml with PBS.

[0227] 4) Take 500 μl of the diluted solution in step (3) and add it to 500 μl of the red blood cell suspension and mix well. The final DTX concentration is 6 μg / ml, 15 μg / ml, and 30 μg / ml.

[0228] 5) Take 500 μl of the diluted solution in step (3) and add it to 500 μl PBS, mix well, and use it as the background tube for each concentration in the experimental group.

[0229] 6) Place all microcentrifuge tubes in a 37°C water bath. After 2 hours, remove the tubes and centrifuge at 3000 g for 10 minutes. Add 200 μl of the supernatant to a 96-well plate and read the absorbance at 540 nm using a microplate reader.

[0230] 7) Calculate the hemolysis rate of erythrocytes using the following formula: Hemolysis rate of erythrocytes = [(absorbance of experimental group - absorbance of background tube) / absorbance of positive control] × 100%.

[0231] Table 2: Comparison of red blood cell hemolysis rate

[0232] Example 3: Lung-targeted docetaxel fat body is more effective in inhibiting lung cancer than docetaxel injection

[0233] Preliminary results showed that the docetaxel adiposomes prepared in Example 2 were stable, safer than docetaxel injection, and more effective than albumin-paclitaxel. However, they lacked targeting, so we further constructed lung-targeted docetaxel adiposomes (Lu-DTX-Ad) and conducted targeting and efficacy validation.

[0234] The specific process for constructing lung-targeted docetaxel fat bodies is as follows.

[0235] 1) Neutral lipids: Mix fish oil and tricaprylin (8:0 TAG) in a volume ratio of 1 / 1.

[0236] 2) Dissolve docetaxel (DTX) in anhydrous ethanol to a concentration of 20 mg / ml.

[0237] 3) 200 μl of the neutral lipid prepared in different volume ratios was taken and added to 200 μl of docetaxel solution, and the ethanol was blown dry with high-purity nitrogen to obtain neutral lipid containing DTX.

[0238] 4) LTA-P33 (peptide sequence: MELTIFILRLAIYILTFPLYLLNFLGLWCRGDK, SEQ ID NO: 4) was dissolved in a mixture of methanol and chloroform (1:1, v / v) to a final concentration of 0.5 mg / ml.

[0239] 5) Mix 20 μl of the solution from step 4) and 80 μl of 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine solution (containing 2 mg DOPC) to a total volume of 100 μl in a microcentrifuge tube, and blow dry the solvent with high-purity nitrogen gas.

[0240] 5) Add 100 μl of PBS and 5 μl of the neutral lipid containing DTX prepared in step 3) to a microcentrifuge tube and vortex for 4 minutes (vortex for 10 seconds, rest for 10 seconds) (in actual application, vortex for 3-7 minutes, vortexing conditions are 3000-4000 rpm, and 4000 rpm is used in this example) to obtain a milky white lipid mixture 1. The lipid mixture 1 is centrifuged at 1000 g for 5 minutes (in actual application, centrifugation at 800-1200 g for 3-7 minutes is acceptable). After centrifugation, the liquid phase system exhibits two stratifications. The lower milky white solution is collected by extraction and vortexed to obtain a milky white lipid mixture 2.

[0241] 6) The lipid mixture 2 obtained in step 5) was centrifuged at 20,000 g for 5 minutes (in actual application, 18,000-22,000 g for 3-7 minutes is acceptable). After centrifugation, the precipitate at the bottom of the microcentrifuge tube was removed and vortexed to obtain a milky white lipid mixture 3.

[0242] 7) The lipid mixture 3 obtained in step 6) was centrifuged at 1000 g for 5 minutes (in actual application, 800-1200 g for 3-7 minutes can be used). After centrifugation, the liquid phase system showed two layers. The lower milky white solution was collected by extraction and vortexed to obtain a milky white lipid mixture 4, which was the final docetaxel adiposome with LTA-P33. Since LTA-P33 can specifically recognize the highly expressed protein Neuropilin-1 in the lungs, the docetaxel adiposome with LTA-P33 was recorded as a lung-targeted docetaxel adiposome (Lung-Targeted Docetaxel Adiposome, Lu-DTX-Ad), and targeting verification and efficacy verification were performed.

[0243] The prepared fluorescently labeled lung-targeted docetaxel adipocytes (Lu-DTX-Ad) and normal saline were injected into mice through the tail vein, respectively. Three hours after the injection, the various tissues were dissected to identify the tissue distribution of Lu-DTX-Ad. Lu-DTX-Ad was almost entirely enriched in the lung tissue (Figure 9A), indicating that we have successfully constructed lung-targeted docetaxel adipocytes and further evaluated its effectiveness in treating lung cancer. We injected 4T1-Luc2 cells into mice through the tail vein. On the fourth day after the injection of the cells, the signal of lung tumor cells was detected by luciferase activity. The mice were randomly divided into three groups: normal saline group (n=8), docetaxel injection group (n=7) and lung-targeted docetaxel adipocyte group (n=8). The dosage was equivalent to 15 mg / kg of docetaxel, administered intravenously once a week for a total of two times. During the dosing period, changes in lung tumor volume were measured in mice. As shown in the figure, the lung tumor signal in the docetaxel injection-treated group was smaller than that in the saline group, with an average tumor inhibition rate of 45.3%. The lung tumor signal in the docetaxel fat body-treated group was minimal, with an average tumor inhibition rate of 79.8% (Figure 9, B). These results demonstrate that the constructed lung-targeted docetaxel fat body is more effective in inhibiting lung cancer than the clinical drug docetaxel injection.

[0244] Example 4 Liver-targeted docetaxel fat bodies are more effective in inhibiting liver cancer than docetaxel injection

[0245] We further constructed liver-targeted docetaxel adiposome (Lv-DTX-Ad) and performed targeting and efficacy verification.

[0246] The process for constructing liver-targeted docetaxel fat bodies is as follows:

[0247] 1) Non-targeted docetaxel fat bodies were prepared according to the method in Example 2.

[0248] 2) Express and purify the recombinant protein ApoE through a prokaryotic system.

[0249] 3) Take 70 μl of the untargeted docetaxel adiposomes prepared in step 1) (OD600 = 1.5) and add the ApoE protein obtained in step 2) to a final concentration of 0.3 μg / μl. Incubate at room temperature for 1 hour, gently vortexing the tube every 10 minutes. Then, purify the ApoE-containing docetaxel adiposomes by centrifugation as follows: centrifuge at 20,000 g for 5 minutes at room temperature until the liquid phase separates into two layers. Remove the lower layer, retain the upper layer, and resuspend the upper layer in 100 μl of PBS buffer three times. Finally, obtain the ApoE-containing docetaxel adiposomes. Because ApoE can specifically recognize the low-density lipoprotein receptor (LDLR), a protein highly expressed in the liver, the ApoE-containing docetaxel adiposomes are designated liver-targeted docetaxel adiposomes (Lv-DTX-Ad). Targeting and efficacy verification are then performed.

[0250] Method for expressing and purifying recombinant protein ApoE in prokaryotic system:

[0251] The ApoE-Flag gene (a fusion gene of ApoE and Flag obtained by removing the ApoE signal peptide sequence and fusing the Flag tag to the C-terminus of ApoE, SEQ ID NO: 1) was constructed into the vector pET28a-SMT3, and the resulting recombinant vector with the correct sequence was designated pET28a-SMT3-ApoE-Flag.

[0252] The constructed recombinant vector pET28a-SMT3-ApoE-Flag was introduced into Escherichia coli Rosetta to obtain recombinant bacteria, designated E-pET28a-SMT3-ApoE-Flag. pET28a-SMT3-ApoE-Flag was inoculated into LB liquid medium containing kanamycin and cultured at 37°C with a shaker at 200 rpm. When the bacterial solution reached an OD600 of 0.6, isopropylthiogalactopyranoside (IPTG) was added to the culture system to a final concentration of 0.4 mM. The culture was then induced at 16°C for 24 hours. The induced culture was collected, resuspended in buffer A, and then disrupted using a JG-1A high-pressure cell disruptor to obtain a bacterial lysate. The resulting cell lysate was ultracentrifuged at 30,000 g for 60 minutes, and the supernatant was collected. 50 μl of the supernatant was added to an equal volume of 2xSample Buffer. The resulting mixture was designated as Sample 1 (supernatant fraction). The remaining supernatant was incubated with Chelating Sepharose Fast Flow, a nickel-chelated filler. After incubation at 4°C for 2 h, the sample was transferred to a 4 ml column. The flow-through was collected (i.e., the flow-through). 50 μl of the flow-through was added to an equal volume of 2xSample Buffer. The resulting mixture was designated as Sample 2 (the flow-through fraction). Nonspecific bands were first washed with the filler in the column by resuspending it in 40 mM imidazole. The flow-through was collected. 50 μl of the wash was added to an equal volume of 2xSample Buffer. The resulting mixture was designated as Sample 3 (40 mM fraction). The target protein was then eluted by resuspending the filler in 500 mM imidazole. The flow-through was collected. 50 μl of the eluate was added to an equal volume of 2xSample Buffer. The resulting mixture was designated as Sample 4 (500 mM fraction). Finally, take another eluate and add Ulp1 to it to enzymatically cleave the SMT3 tag at the nitrogen end of the target protein, and add the enzymatic cleavage product to the column again for reverse hanging to remove the SMT3 tag. Collect the effluent to obtain the recombinant protein ApoE solution. Take 50μl of the recombinant protein ApoE solution and add 2xSample Buffer. The resulting mixture is used as sample 5 (ApoE component). The resulting mixture is placed in a dialysis bag, and then the dialysis bag is placed in a beaker containing 100 times the volume of buffer A of the dialysis bag and dialyzed overnight. Take 50μl of the solution in the dialysis bag and add an equal volume of 2xSample Buffer. The resulting mixture is used as sample 6 (dialysis component). The above samples 1-6 are analyzed by SDS-PAGE and then subjected to staining analysis and identification.Lanes 1 and 2 contained numerous nonspecific bands, and the target protein, ApoE-Flag, was primarily concentrated in sample 1. Lane 4 contained the fusion protein at a molecular weight of 55 kDa, while lanes 5 and 6 contained the recombinant ApoE-Flag protein, cleaved from the HIS-SMT3 tag, at a molecular weight of 35 kDa. These results demonstrate that the above method successfully generated the prokaryotic recombinant ApoE-Flag protein, referred to as ApoE in this experiment (Figure 10A).

[0253] ApoE nucleotide sequence (removing the ApoE signal peptide sequence and fusing the Flag tag to the C-terminus of ApoE)

[0254] ApoE amino acid sequence (removing the ApoE signal peptide sequence and fusing the Flag tag to the C-terminus of ApoE)

[0255] The morphology of the obtained liver-targeted docetaxel adiposomes (Lv-DTX-Ad) was observed under an optical microscope. Lv-DTX-Ad was a spherical structure of uniform size and could be stained red with the neutral lipid-specific dye LipidTox Red (Figure 10B). Western blot analysis revealed that LDLR was highly expressed in the liver of mice, as well as in the hepatocellular carcinoma cell lines HepG2 and Hepa1-6, while expression was very low in the embryonic kidney epithelial cell line HEK293 (Figure 10C). The prepared fluorescently labeled liver-targeted docetaxel adiposomes were injected into mice via the tail vein. After injection, tissues were dissected and analyzed at different times to identify the tissue distribution of the adiposomes. At different injection times, the liver-targeted docetaxel adiposomes were enriched in the liver. 24 hours after injection, the liver-targeted docetaxel adiposomes still had a strong signal in the liver (Figure 10D). This indicates that we have successfully constructed liver-targeted docetaxel fat bodies and further evaluated their efficacy and safety in the treatment of liver cancer.

[0256] We injected Hepa1-6 cells, a mouse liver cancer cell line, orthotopically into the livers of mice. Three days after cancer cell injection, the mice were randomly divided into three groups, each containing five mice: a saline group, a docetaxel injection group, a docetaxel adipocyte group, and a liver-targeted docetaxel adipocyte group. Docetaxel equivalent to 15 mg / kg was administered intravenously once a week for three times. Thirty-one days after cancer cell injection, the mice were sacrificed, their livers isolated, and liver tumor size measured. Blood was also collected for liver and kidney function tests. As shown in the figure, compared to the saline group, the liver tumor volume in the docetaxel injection group was significantly smaller, with an average tumor inhibition rate of 47.4%. The liver tumor inhibition rate in the docetaxel adipocyte group was 84.5%, significantly better than that in the docetaxel injection group. The liver-targeted docetaxel adipocyte group achieved the highest inhibition rate, reaching 97.9% (Figure 11A). In addition, the average weight loss of mice in the liver-targeted docetaxel adipocyte treatment group was 7%, the average weight loss of mice in the docetaxel adipocyte treatment group was 14.4%, and the average weight loss of mice in the docetaxel injection treatment group was 38.9% (Figure 11B). At the same time, the liver function inflammation indicator AST (aspartate aminotransferase) in the liver-targeted docetaxel adipocyte treatment group was significantly lower than that in the docetaxel injection treatment group. Urea (urea) and creatinine (CRE) indicators had no effect, indicating that there were no toxic side effects on renal function. These results show that the effectiveness of liver-targeted docetaxel adipocytes and docetaxel adipocytes in inhibiting liver cancer is superior to the clinical drug docetaxel injection, and they also have advantages in safety.

[0257] Example 5 The effect of hematological tumor cells targeting docetaxel fat bodies is better than docetaxel injection

[0258] We further constructed H929-Targeted Docetaxel Adiposome (H929-DTX-Ad) targeting hematologic malignancy cell line H929 and performed targeting and efficacy verification.

[0259] 1. Preparation of biotin-labeled fat bodies

[0260] Materials: 18:1(Δ9-Cis)PC, 18:1(DOPC) without biotin; 18:1Biotinyl Cap PE, 18:1(Δ9-Cis)PC, and 18:1(Δ9-Cis)PC; and 18:1(Δ9-Cis)PC, all purchased from Sigma.

[0261] (1) Dissolve the above-mentioned DOPC in chloroform to a final concentration of 25 mg / ml. Dissolve the above-mentioned Bio-PE in chloroform to a final concentration of 0.04 mg / ml.

[0262] (2) Add 70 μl of DOPC and 20 μl of Bio-PE to a microcentrifuge tube, vortex mix thoroughly, and blow dry the solvent with high-purity nitrogen gas.

[0263] (3) Add 100 μl of phosphate buffered saline (PBS) and 5 mg of triglyceride (TAG) or other neutral lipids to a microcentrifuge tube and vortex for 5 min (vortex for 10 s, stop for 5 s) to obtain a milky white lipid mixture 1 (i.e., the initial preparation component). Centrifuge the lipid mixture 1 at 1000 g for 5 min, remove the upper white band, and collect the lower layer as mixture 2.

[0264] (4) The lipid mixture 2 is centrifuged at 20,000 g for 5 min (in actual application, 18,000-22,000 g for 3-7 min is acceptable). After centrifugation, the sediment at the bottom of the tube is removed, and the remaining emulsion in the tube is resuspended to obtain a milky white lipid mixture 3.

[0265] (5) The lipid mixture 3 was centrifuged at 1000 g for 5 min. After centrifugation, the upper white band was removed and the remaining emulsion in the tube was resuspended to obtain a milky white lipid mixture 4, which is the final biotin-labeled fat body.

[0266] 2. Preparation of hematologic tumor cell-targeted adipocytes

[0267] Take 30ul of the biotin-labeled fat body prepared as above, then add 20ul of avidin-labeled BCMA antibody (purchased from Jingtiancheng Biotechnology Co., Ltd., the amino acid sequence is shown in SEQ ID NO: 3) to a final concentration of 0.25mg / ml, incubate at room temperature for 1 hour, and centrifuge the lipid mixture at 21000g for 6min. After centrifugation, remove the sediment at the bottom of the tube, and suspend the remaining emulsion in the tube to obtain a milky white lipid mixture. The lipid mixture was centrifuged at 1000g for 6min. After centrifugation, remove the upper white band and resuspend the remaining emulsion in the tube. Repeat the above steps again for a total of three times to obtain the final fat body with BCMA antibody. As shown in Figure 12 A, the amount of BCMA antibody added remains unchanged, and the content of the fat body is gradually increased. It can be seen that since the BCMA antibody is recruited to the surface of the fat body by the avidin-biotin method, the molecular weight of the BCMA antibody gradually migrates upward (as shown by arrow 1), indicating that we have successfully prepared fat bodies carrying BCMA antibodies, that is, blood tumor cell-targeted fat bodies.

[0268] BCMA antibody amino acid sequence:

[0269] 3. Hematologic tumor cell targeting: Adipocyte targeting of hematologic tumor cells H929

[0270] Relapsed myeloma is caused by hematologic malignancy cells H929, which highly express the protein BCMA (Tumor necrosis factor receptor superfamily member 17). Western blot revealed that only hematologic malignancy cells H929 highly expressed BCMA protein, while another hematologic malignancy cell line, Raji, and human embryonic kidney epithelial cells HEK293 did not express BCMA protein (Figure 12B). To further explore the targeting of hematologic malignancy cell-targeted adipocytes, the following groups were used: a blank group (no treatment), a non-targeted adipocyte treatment group, a hematologic malignancy cell-targeted adipocyte group (adipocytes with BCMA antibodies), and an inhibition-targeted adipocyte group (adipocytes with BCMA antibodies were blocked by adding BCMA protein). All adipocyte samples were fluorescently labeled. The above samples were collected for flow cytometry to detect the fluorescence signal in the cells. Among them, the peak of the hematologic tumor cell-targeted fat body group shifted significantly to the right (peak 3 in C in Figure 12), indicating that H929 cells phagocytosed more fat bodies, while the peaks of the non-targeted fat body treatment group and the inhibition of targeted fat body group did not shift to the right (peak 2 and peak 4 in C in Figure 12). The statistical results also showed that the fluorescence signal of the hematologic tumor cell-targeted fat body group was the strongest (column 3 in C in Figure 12), which was significantly higher than that of the non-targeted fat body treatment group (column 2 in C in Figure 12), while the inhibition of targeted fat body group inhibited the endocytosis of fat bodies by H929 cells. Fluorescence observation was performed at the same time, and consistent with the results of flow cytometry, H929 cells had the strongest endocytosis of hematologic tumor cell-targeted fat bodies, and the addition of BCMA protein inhibited the endocytosis of H929 cells by hematologic tumor cell-targeted fat bodies (D in Figure 12). The above results show that the prepared hematologic tumor cell-targeted fat bodies successfully targeted the hematologic tumor cells H929.

[0271] 4. Hematologic malignancy cell targeting of docetaxel fat bodies is more effective than docetaxel injection

[0272] Since the above experiments proved that adipocytes targeting hematological tumor cells H929 were successfully constructed, we further constructed adipocytes containing docetaxel that can target hematological tumor cells. The steps are as follows:

[0273] 1) Neutral lipids: Mix fish oil and tricaprylin (8:0 TAG) in a volume ratio of 1 / 1.

[0274] 2) Dissolve docetaxel (DTX) in anhydrous ethanol to a concentration of 20 mg / ml.

[0275] 3) 200 μl of the neutral lipid prepared in different volume ratios was taken and added to 200 μl of docetaxel solution, and the ethanol was blown dry with high-purity nitrogen to obtain neutral lipid containing DTX.

[0276] 4) Add 70ul of DOPC and 20ul of Bio-PE to a microcentrifuge tube, vortex mix thoroughly, and blow dry the solvent with high-purity nitrogen gas.

[0277] 5) Add 100 μl of PBS and 5 μl of the neutral lipid containing DTX prepared in step 3) to a microcentrifuge tube and vortex for 4 minutes (vortex for 10 seconds, rest for 10 seconds) (in actual application, vortex for 3-7 minutes, vortexing conditions are 3000-4000 rpm, and 4000 rpm is used in this example) to obtain a milky white lipid mixture 1. The lipid mixture 1 is centrifuged at 1000 g for 5 minutes (in actual application, centrifugation at 800-1200 g for 3-7 minutes is acceptable). After centrifugation, the liquid phase system exhibits two stratifications. The lower milky white solution is collected by extraction and vortexed to obtain a milky white lipid mixture 2.

[0278] 6) The lipid mixture 2 obtained in step 5) was centrifuged at 20,000 g for 5 minutes (in actual application, 18,000-22,000 g for 3-7 minutes is acceptable). After centrifugation, the precipitate at the bottom of the microcentrifuge tube was removed and vortexed to obtain a milky white lipid mixture 3.

[0279] 7) The lipid mixture 3 obtained in step 6) was centrifuged at 1000 g for 5 min (in actual application, 800-1200 g for 3-7 min can be used). After centrifugation, the liquid phase system showed two layers. The lower milky white solution was collected by extraction and vortexed to obtain a milky white lipid mixture 4.

[0280] 8) 30ul of lipid mixture 4 obtained in step 7) was then added with 20ul of avidin-labeled BCMA antibody at a final concentration of 0.25mg / ml, incubated at room temperature for 1 hour, and the lipid mixture was centrifuged at 21000g for 6min. After centrifugation, the sediment at the bottom of the tube was removed, and the remaining emulsion in the tube was suspended to obtain a milky white lipid mixture 5. The lipid mixture was centrifuged at 1000g for 6min. After centrifugation, the upper white band was removed and the remaining emulsion in the tube was resuspended. The above steps were repeated three times to obtain hematologic tumor cell-targeted docetaxel fat bodies.

[0281] We further tested whether the hematologic tumor cell-targeted docetaxel fat bodies have biological activity and can kill tumor cells. Human hematologic tumor cells H929 were treated with commercial docetaxel injection, the non-targeted docetaxel fat bodies prepared in Example 2, and hematologic tumor cell-targeted docetaxel fat bodies, respectively. After treating the cells with the same concentration of 20 ng / ml of DTX for 2 hours, 4 hours, and 16 hours, the culture medium was removed, and then drug-free culture medium was added to measure cell survival. The results showed that after 2 hours and 4 hours of treatment, the cell survival rate of the hematologic tumor cell-targeted docetaxel fat body treatment was better than that of docetaxel injection and non-targeted docetaxel fat body, indicating that the effect of hematologic tumor cell-targeted docetaxel fat body in killing H929 cancer cells in a short time was better than that of docetaxel injection and non-targeted docetaxel fat body (Figure 13).

[0282] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A docetaxel fat body preparation comprising: A monomolecular phospholipid membrane and docetaxel and neutral lipids encapsulated in the monomolecular phospholipid membrane.

2. The preparation according to claim 1, characterized in that The monomolecular phospholipid membrane comprises one or more of phospholipids, functional polar lipids and cationic lipids; The phospholipid is selected from one or more of 2-bis-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine, egg yolk lecithin, soybean lecithin, dioleoylphosphatidylethanolamine, distearoylphosphatidylcholine, egg yolk lecithin, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidic acid, sodium distearoylphosphatidylglycerol, dimyristoylphosphatidylcholine, 1-stearoyl-lysophosphatidylcholine, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000, phosphatidylethanolamine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, cardiolipin and sphingomyelin; The functional polar lipid is selected from one or more of polyethylene glycol-modified sterols, biotin-modified sterols, amino acid-modified sterols, polypeptide-modified sterols, polysaccharide-modified sterols, nucleic acid-modified sterols, polyethylene glycol-modified phospholipids, biotin-modified phospholipids, amino acid-modified phospholipids, polypeptide-modified phospholipids, polysaccharide-modified phospholipids and nucleic acid-modified phospholipids; The cationic lipid is selected from one or more of (2,3-dioleoyl-propyl)-trimethylammonium-chloride, (2,3-dioleoyl-propyl)-trimethylamine, 2,3-dioleoyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylamine hydrochloride, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride.

3. The preparation according to claim 1, characterized in that The neutral lipid is selected from one or more of fish oil, corn oil, tricaprylin, triolein, retinol ester, wax ester, sterol ester, sterol ester, castor oil, sunflower oil, soybean oil, peanut oil, clove oil, simethicone, cinnamon oil, tea oil, liquid paraffin, star anise oil, mixed fatty acid glycerides (stearin), hydrogenated vegetable oil, refined olive oil, refined olive oil, retinol ester and fat-soluble vitamins.

4. The preparation according to any one of claims 1 to 3, characterized in that The monomolecular phospholipid membrane includes 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine; The neutral lipids are fish oil and tricaprylin.

5. The preparation according to claim 4, characterized in that The volume ratio of fish oil to tricaprylin in the neutral fat is 1:(0.1-10).

6. The preparation according to any one of claims 1 to 5, characterized in that Also included are targeting molecules that target and recognize organs, tissues, or cells.

7. The preparation according to claim 6, characterized in that The organs, tissues or cells are from the human body or animal body.

8. The preparation according to claim 6 or 7, characterized in that The targeting molecule is: The targeting molecule is embedded in a single molecule phospholipid membrane, or is connected to phospholipids through avidin-biotin interaction, or is connected to a substance that specifically targets phospholipids, or a combination of the above methods.

9. The preparation according to claim 6 or 7, characterized in that The targeting molecule is: At least one of LTA-P33, ApoE, BCMA antibody, Nrp-B, Trf-B, LDLR-B, ErbB2-B, CXCR4-B, GRP78-B or Soma-B.

10. The preparation according to any one of claims 6 to 9, characterized in that One or more of the phospholipids, functional polar lipids and cationic lipids in the monomolecular phospholipid membrane is labeled with biotin or streptavidin.

11. The preparation according to claim 9, characterized in that The targeting molecule is LTA-P33; The neutral lipids are fish oil and tricaprylin; The monomolecular phospholipid membrane is 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine; The volume ratio of the fish oil to tricaprylin is 1:(0.1-10).

12. The preparation according to claim 9, characterized in that The targeting molecule is ApoE; The neutral lipids are fish oil and tricaprylin; The monomolecular phospholipid membrane is 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine; The volume ratio of the fish oil to tricaprylin is 1:(0.1-10).

13. The preparation according to claim 9, characterized in that The targeting molecule is a BCMA antibody, and the BCMA is labeled with streptavidin; The neutral lipid is triolein; The monomolecular phospholipid membrane is a phospholipid labeled with 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine and biotin; The mass ratio of the 2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine and the biotin-labeled phospholipid is (0-200):(0.05-200).

14. A method for preparing the preparation according to any one of claims 1 to 5, comprising: Step 1: mixing a docetaxel solution and a neutral lipid, and removing the solvent to obtain a neutral lipid containing docetaxel; Step 2: The neutral lipids and phospholipids containing docetaxel are mixed, and the docetaxel-loaded fat bodies are obtained after repeated vortexing and centrifugation.

15. A method for preparing the preparation according to any one of claims 6 to 13, comprising: Step A: mixing a docetaxel solution and a neutral lipid, and removing the solvent to obtain a neutral lipid containing docetaxel; Step B: mixing the targeting molecule solution with the phospholipid, and removing the solvent to obtain the phospholipid containing the targeting molecule; Step C: The neutral lipid containing docetaxel and the phospholipid containing the targeting molecule are mixed, and the docetaxel-encapsulated fat body is obtained after repeated vortexing and centrifugation.

16. A method for preparing the preparation according to any one of claims 6 to 13, comprising: Step a: mixing a docetaxel solution and a neutral lipid, and removing the solvent to obtain a neutral lipid containing docetaxel; Step b: mixing the neutral lipid containing docetaxel with a buffer and phospholipids, and repeatedly vortexing and centrifuging; Step c: mixing with targeting molecules and incubating to obtain docetaxel-loaded fat bodies.

17. The preparation method according to any one of claims 14 to 16, characterized in that In the docetaxel solution, the solvent is an organic solvent, and the organic solvent is any one of anhydrous ethanol, chloroform, methanol, or a combination of two or more thereof; In the targeting molecule solution, the solvent is an organic solvent, and the organic solvent is methanol and anhydrous ethanol, chloroform, benzene, toluene, xylene, butanol, isopropanol, ether, acetone, cyclohexanone, A mixture of at least one of methyl isobutyl ketone, ethyl acetate, butyl acetate, cyclohexanone or petroleum ether; The buffer solution is PBS buffer solution, HEPES buffer solution, sucrose solution, NaCl solution, KCl solution or MgCl2 solution.

18. The preparation method according to any one of claims 14 to 16, characterized in that The repeated vortexing and centrifugation include: After mixing, the mixture was vortexed to obtain a mixture 1. The mixture 1 is centrifuged to collect the lower layer solution, and then vortexed again to obtain a mixture 2; After the mixture 2 is centrifuged to remove the precipitate, it is vortexed again to obtain the mixture 3; The mixture 3 is centrifuged to collect the lower layer solution, which is vortexed again to obtain the mixture 4 containing the fat body.

19. Use of the preparation according to any one of claims 1 to 13 or the preparation obtained by the method according to any one of claims 14 to 18 in the preparation of drugs or vaccines for preventing and treating tumors.

20. The use according to claim 19, characterized in that The tumors include: lung cancer, kidney cancer, laryngeal cancer, liver cancer, muscle tissue cancer, blood tumors, bone cancer, brain cancer, breast cancer, neck cancer, oral or nasal mucosal cancer, bladder cancer, central nervous system cancer, cervical cancer, head and neck cancer, colon cancer, endometrial cancer, external genital cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, genitourinary tract cancer, head cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, melanoma, testicular cancer and / or thyroid cancer.

21. A medicine or vaccine comprising the preparation according to any one of claims 1 to 13 or the preparation prepared by the method according to any one of claims 14 to 18.

22. The drug or vaccine according to claim 21, characterized in that It also includes an effective amount of a tumor suppressor; The tumor suppressors include: cisplatin, carboplatin, oxaliplatin, 5-fluorouracil (5-FU), methotrexate, daunorubicin, dactinomycin-D, irinotecan (CPT-11), mitoxantrone, estramustine, vincristine, dexamethasone, prednisone, lomustine, methotrexate, pirarubicin, doxorubicin, gemcitabine, quizartinib or bevacizumab.

23. A method for preventing and treating tumors, comprising administering the drug or vaccine according to claim 21 or 22.

24. The method according to claim 23, wherein The subject of the method is a human.

25. The method according to claim 23, characterized in that The subject of the method is a primate or a non-primate mammal.

Images