本申请要求于2024年03月05日提交中国专利局、申请号为202410251555.7、发明名称为“新藤黄酸脂肪体及其制备方法与应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims priority to the Chinese patent application filed with the China Patent Office on March 5, 2024, with application number 202410251555.7 and invention name “New Garcinia Acid Fat Body and Its Preparation Method and Application”, the entire contents of which are incorporated herein by reference.
本发明涉及药物技术领域，尤其涉及新藤黄酸脂肪体制剂及其制备方法和应用。The present invention relates to the field of pharmaceutical technology, in particular to a new gambogic acid fat body preparation and a preparation method and application thereof.
新藤黄酸(Neogambogic acid,NGA)是中药藤黄中的主要成分之一，具有抗氧化、抗炎、抗癌和抗菌等作用。越来越多的研究表明，新藤黄酸可以抑制乳腺癌，肺癌，前列腺癌，胃癌，和头颈癌等癌症的发生和发展，主要通过诱导癌细胞凋亡、自噬、细胞周期停滞等多种机制。Neogambogic acid (NGA), a major component of the traditional Chinese medicine Garcinia cambogia, possesses antioxidant, anti-inflammatory, anticancer, and antibacterial properties. A growing body of research suggests that NGA can inhibit the development and progression of breast, lung, prostate, gastric, and head and neck cancers, primarily through inducing apoptosis, autophagy, and cell cycle arrest.
新藤黄酸是一种疏水性小分子，logP(油水分配系数)为6.3，在水中的溶解度极低，小于0.0005mg/mL。其疏水性严重影响其吸收和生物利用度，同时会导致不同程度的毒副作用，因此严重限制其临床转化。Neogamoic acid is a hydrophobic small molecule with a logP (oil-water partition coefficient) of 6.3 and an extremely low solubility in water, less than 0.0005 mg/mL. Its hydrophobicity significantly affects its absorption and bioavailability, while also leading to varying degrees of toxic side effects, thus severely limiting its clinical application.
为了提高新藤黄酸的溶解度、有效性和安全性，许多纳米载体制剂应用于包载新藤黄酸，但是这些方法也存在一些缺点，如脂质体具有一个亲水核心，只有其磷脂双分子层之间是疏水的环境可以用于携带新藤黄酸，但是其包载量较低。此外，携带新藤黄酸的脂质体的稳定性较差，容易发生融合、破裂或药物泄露，这可能影响其在体内的分布和药物的释放。同样的，聚合物载体包载新藤黄酸的量也较低。另外，聚合物PEG和PLGA在体内非天然存在，会使机体产生相应的抗体来抵抗其作用。固体脂质纳米颗粒由于在制备过程中需要高温高压等步骤，容易破坏新藤黄酸分子的结构。In order to improve the solubility, efficacy and safety of neogamoic acid, many nanocarrier preparations have been used to encapsulate neogamoic acid, but these methods also have some disadvantages. For example, liposomes have a hydrophilic core, and only the hydrophobic environment between their phospholipid bilayers can be used to carry neogamoic acid, but their encapsulation capacity is low. In addition, the stability of liposomes carrying neogamoic acid is poor, and they are prone to fusion, rupture or drug leakage, which may affect its distribution in the body and drug release. Similarly, the amount of neogamoic acid encapsulated by polymer carriers is also low. In addition, polymers PEG and PLGA do not exist naturally in the body, which will cause the body to produce corresponding antibodies to resist their effects. Solid lipid nanoparticles require high temperature and high pressure steps during the preparation process, which easily destroys the structure of the neogamoic acid molecule.
新型纳米颗粒脂肪体具有一个中性脂的疏水核心，由单分子磷脂膜包裹的纳米小球，与天然存在的脂滴和脂蛋白结构类似，可以高效溶解和包载疏水性小分子化合物，此外脂肪体的成分在机体内天然存在，具有很好的生物相容性，另外制备方法简单、高效。倘若将脂肪体应用于担载新藤黄酸或其他疏水性药物，则有望提高药物的溶解度，改善生物利用度，提高有效性和安全性，这将为患者的治疗带来极大的便利。The new nanoparticle fat body has a hydrophobic core of neutral lipids and a nanosphere wrapped by a single molecule phospholipid membrane. It is similar to the structure of naturally occurring lipid droplets and lipoproteins and can efficiently dissolve and encapsulate Fat bodies can be used to carry hydrophobic small molecules. Furthermore, the components of fat bodies are naturally present in the body, resulting in excellent biocompatibility. Furthermore, the preparation method is simple and efficient. If fat bodies are used to carry neogamonic acid or other hydrophobic drugs, it is expected to increase drug solubility, improve bioavailability, enhance efficacy and safety, and greatly facilitate patient treatment.
发明内容Summary of the Invention
有鉴于此，本发明要解决的技术问题在于提供新藤黄酸脂肪体制剂及其制备方法和应用，该脂肪体制剂具有良好的生物利用度和安全性，且可以担载靶向分子。In view of this, the technical problem to be solved by the present invention is to provide a new gambogic acid fat body preparation and its preparation method and application, which has good bioavailability and safety and can carry targeting molecules.
本发明提供的新藤黄酸脂肪体制剂包括：单分子磷脂膜和包裹在单分子磷脂膜内部的新藤黄酸及中性脂质。The new gambogic acid fat body preparation provided by the present invention comprises: a single molecule phospholipid membrane and new gambogic acid and neutral lipid wrapped in the single molecule phospholipid membrane.
本发明中，所述中性脂质选自甘油三油酸酯、鱼油、玉米油、三辛酸甘油酯、视黄醇酯、蜡酯、甾醇酯、固醇酯、蓖麻油、葵花籽油、大豆油、花生油、丁香油、西甲硅油、肉桂油、茶油、液状石蜡、八角茴香油、混合脂肪酸甘油酯(硬脂)、氢化植物油、精制橄榄油与脂溶性微生素中的一种或多种。In the present invention, the neutral lipid is selected from one or more of triolein, fish oil, corn oil, tricaprylin, retinol esters, wax esters, sterol esters, sterol esters, 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.
本发明中，所述单分子磷脂膜的膜材包含磷脂或功能性极性脂质中的一种或多种。In the present invention, the membrane material of the monomolecular phospholipid membrane comprises one or more of phospholipids or functional polar lipids.
一些实施例中，所述磷脂选自2-二-(9Z-十八碳烯酰基)-sn-甘油-3-磷酸胆碱(DOPC)、蛋黄卵磷脂、大豆磷脂、二油酰磷脂酰乙醇胺、二硬脂酰磷脂酰胆碱、二棕榈酰磷脂酰胆碱、二棕榈酰磷脂酸、二硬脂酰磷脂酰甘油钠、二肉豆蔻酰磷脂酰胆碱、1-硬脂酰-溶血磷脂酰胆碱、1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000、培化磷脂酰乙醇胺、磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰肌醇、磷脂酰丝氨酸、磷脂酰甘油、磷脂酸、心磷脂与鞘磷脂中的一种或多种；In some embodiments, 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, 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.
一些实施例中，所述功能性极性脂选自聚乙二醇修饰固醇、生物素修饰固醇、氨基酸修饰固醇、多肽修饰固醇、多糖修饰固醇、核酸修饰固醇、聚乙二醇修饰磷脂、生物素修饰磷脂、氨基酸修饰磷脂、多肽修饰磷脂、多糖修饰磷脂与核酸修饰磷脂中的一种或多种。In some embodiments, 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.
为了更好的包载新藤黄酸，本发明对单分子磷脂膜的膜材进行了优化和筛选。In order to better encapsulate neogambogic acid, the present invention optimizes and screens the membrane materials of the single-molecule phospholipid membrane.
一些实施例中，所述磷脂选自：蛋黄卵磷脂、大豆磷脂或DOPC。中性脂选自：三油酸甘油酯、葵花籽油或大豆油。In some embodiments, the phospholipid is selected from egg yolk lecithin, soybean lecithin, or DOPC. The neutral lipid is selected from triolein, sunflower oil, or soybean oil.
一些具体实施例中：所述单分子磷脂膜的膜材为蛋黄卵磷脂，所述中性脂质为三油酸甘油酯。In some specific embodiments, the membrane material of the monomolecular phospholipid membrane is egg yolk lecithin, and the neutral lipid is triolein.
或者，所述单分子磷脂膜的膜材为蛋黄卵磷脂，所述中性脂质为葵花籽油。Alternatively, the membrane material of the monomolecular phospholipid membrane is egg yolk lecithin, and the neutral lipid is sunflower oil.
或者，所述单分子磷脂膜的膜材为蛋黄卵磷脂，所述中性脂质为大豆油。Alternatively, the membrane material of the monomolecular phospholipid membrane is egg yolk lecithin, and the neutral lipid is soybean oil.
或者，所述单分子磷脂膜的膜材为大豆磷脂，所述中性脂质为三油酸甘油酯。Alternatively, the membrane material of the monomolecular phospholipid membrane is soybean lecithin, and the neutral lipid is triolein.
或者，所述单分子磷脂膜的膜材为大豆磷脂，所述中性脂质为葵花籽油。Alternatively, the membrane material of the monomolecular phospholipid membrane is soybean lecithin, and the neutral lipid is sunflower oil.
或者，所述单分子磷脂膜的膜材为大豆磷脂，所述中性脂质为大豆油。Alternatively, the membrane material of the monomolecular phospholipid membrane is soybean lecithin, and the neutral lipid is soybean oil.
或者，所述单分子磷脂膜的膜材为DOPC，所述中性脂质为三油酸甘油酯。Alternatively, the membrane material of the monomolecular phospholipid membrane is DOPC, and the neutral lipid is triolein.
或者，所述单分子磷脂膜的膜材为DOPC，所述中性脂质为葵花籽油。Alternatively, the membrane material of the monomolecular phospholipid membrane is DOPC, and the neutral lipid is sunflower oil.
或者，所述单分子磷脂膜的膜材为DOPC，所述中性脂质为大豆油。Alternatively, the membrane material of the monomolecular phospholipid membrane is DOPC, and the neutral lipid is soybean oil.
本发明中，所述单分子磷脂膜还包括阳离子脂质。本发明实验表明，在膜材中加入阳离子脂质，能够进一步提高新藤黄酸脂肪体制剂的生物利用度，提高抗肿瘤活性，扩大新藤黄酸的适用范围，并且能够确保其安全性。本发明中，对阳离子脂质的种类不做限定，只要带有正电荷的脂质都能够实现这种效果上的提升。一些实施例中，所述阳离子脂质选自DOTAP((2,3-二油酰基-丙基)-三甲胺(氯盐))、DODAP((Z)-3-(二甲基氨基)丙烷-1,2-二基二油酸酯)、DODMA(1,2-二油醇-3-二甲基氨基-丙烷)、DOTMA(1,2-双十八烯氧基-3-甲基铵丙烷(氯盐))、D-Lin-MC3-DMA(4-(N,N-二甲基氨基)丁酸(二亚油基)甲酯)中的一种或多种。In the present invention, the monomolecular phospholipid membrane also includes a cationic lipid. Experiments in the present invention have shown that the addition of cationic lipids to the membrane material can further improve the bioavailability of the new gambogic acid fat body preparation, enhance anti-tumor activity, expand the scope of application of new gambogic acid, and ensure its safety. In the present invention, the type of cationic lipid is not limited; as long as it is a lipid with a positive charge, this improvement in effect can be achieved. In some embodiments, the cationic lipid is selected from one or more of DOTAP ((2,3-dioleoyl-propyl)-trimethylamine (chloride)), DODAP ((Z)-3-(dimethylamino)propane-1,2-diyl dioleate), DODMA (1,2-dioleyl-3-dimethylamino-propane), DOTMA (1,2-dioctadecenyloxy-3-methylammonium propane (chloride)), and D-Lin-MC3-DMA (4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester).
一些实施例中：In some embodiments:
所述单分子磷脂膜的膜材为蛋黄卵磷脂和DOTAP，所述中性脂质为三油酸甘油酯。The membrane materials of the monomolecular phospholipid membrane are egg yolk phosphatidylcholine and DOTAP, and the neutral lipid is triolein.
或者，所述单分子磷脂膜的膜材为蛋黄卵磷脂和DOTAP，所述中性脂质为葵花籽油。Alternatively, the membrane material of the monomolecular phospholipid membrane is egg yolk lecithin and DOTAP, and the neutral lipid is sunflower oil.
或者，所述单分子磷脂膜的膜材为蛋黄卵磷脂和DOTAP，所述中性脂质为大豆油。Alternatively, the membrane material of the monomolecular phospholipid membrane is egg yolk lecithin and DOTAP, and the neutral lipid is soybean oil.
或者，所述单分子磷脂膜的膜材为大豆磷脂和DOTAP，所述中性脂质为三油酸甘油酯。Alternatively, the membrane material of the monomolecular phospholipid membrane is soybean lecithin and DOTAP, and the neutral lipid is triolein.
或者，所述单分子磷脂膜的膜材为大豆磷脂和DOTAP，所述中性脂质为葵花籽油。Alternatively, the membrane materials of the monomolecular phospholipid membrane are soybean lecithin and DOTAP, and the neutral lipid is sunflower oil.
或者，所述单分子磷脂膜的膜材为大豆磷脂和DOTAP，所述中性脂质为大豆油。Alternatively, the membrane materials of the monomolecular phospholipid membrane are soybean lecithin and DOTAP, and the neutral lipid is soybean oil.
或者，所述单分子磷脂膜的膜材为DOPC和DOTAP，所述中性脂质为三油酸甘油酯。Alternatively, the membrane material of the monomolecular phospholipid membrane is DOPC and DOTAP, and the neutral lipid is triolein.
或者，所述单分子磷脂膜的膜材为DOPC和DOTAP，所述中性脂质为葵花籽油。Alternatively, the membrane material of the monomolecular phospholipid membrane is DOPC and DOTAP, and the neutral lipid is sunflower oil.
或者，所述单分子磷脂膜的膜材为DOPC和DOTAP，所述中性脂质为大豆油。Alternatively, the membrane material of the monomolecular phospholipid membrane is DOPC and DOTAP, and the neutral lipid is soybean oil.
进一步的，本发明所述的新藤黄酸脂肪体制剂中，还包括靶向分子，所述靶向分子靶向识别器官、组织或细胞。Furthermore, the new gambogic acid fat body preparation of the present invention also includes a targeting molecule, which targets and recognizes organs, tissues or cells.
本发明中，所述器官、组织或细胞来自人体或动物体；In the present invention, the organs, tissues or cells are from the human body or animal body;
所述器官为内分泌器官、消化器官、循环器官、泌尿器官、生殖器官、运动器官、神经系统和感觉器官。其中内分泌器官包括甲状腺、胰腺。消化器官包括胃、肝脏、胆囊、脾、胰腺、小肠、大肠。呼吸器官包括肺。循环器官包括心脏、血管。泌尿器官包括肾脏、输尿管、膀胱。生殖器官包括子宫、卵巢。运动器官包括肌肉、骨骼。神经系统包括大脑、小脑。感觉器官包括皮肤、眼睛、耳朵。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.
所述组织或细胞来自人体或动物体的肿瘤。所述肿瘤包括：肺癌、肾癌、喉癌、肝癌、肌肉组织癌症、血液瘤、骨癌、脑癌、乳腺癌、颈癌、口腔或鼻黏膜癌、膀胱癌、中枢神经系统癌症、宫颈癌、头颈癌、结肠癌、子宫内膜癌、外生殖器癌、食管癌、胆囊癌、胃肠道癌、泌尿生殖道癌、头癌、卵巢癌、胰腺癌、前列腺癌、皮肤癌、脾癌、小肠癌、大肠癌、胃癌、黑色素瘤、睾丸癌和/或甲状腺癌。The tissue or cell is derived from a tumor in a 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, neck cancer, Cancer of the oral or nasal mucosa, bladder, central nervous system, cervix, head and neck, colon, endometrium, external genitalia, esophagus, gallbladder, gastrointestinal tract, genitourinary tract, head, ovary, pancreas, prostate, skin, spleen, small intestine, large intestine, stomach, melanoma, testicle, and/or thyroid.
所述靶向分子可以嵌入单分子磷脂膜中，也可以通过亲和素-生物素系统与单分子磷脂膜上的磷脂结合，也可以与特异性靶向磷脂的物质连接，从而结合在单分子磷脂膜上，或为上述方式中任意两者或两者以上的组合。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.
本发明中，所述靶向分子为：LTA-P33、ApoE、BCMA抗体、Nrp-B、Trf-B、LDLR-B、ErbB2-B、CXCR4-B、GRP78-B或Soma-B中至少一种。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.
一些实施例中，所述靶向分子上连接生物素，在制备脂肪体的过程中，加入标记有链霉亲和素的磷脂分子。或者所述靶向分子上连接链霉亲和素，在制备脂肪体的过程中，加入标记有生物素的磷脂分子。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.
一些实施例中，所述靶向分子上连接有靶向识别单分子磷脂膜的肽段，其包括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和PF中至少一种。所述靶向分子与靶向识别单分子磷脂膜的肽段可以通过linker连接，也可以不通过linker直接连接，本发明对此不做限定。所述linker为可剪切linker或自剪切linker。所述可剪切linker的氨基酸序列为LEAGCKNFFPRSFTSCGSLE，所述自剪切linker为P2A、T2A或者E2A。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.
本发明中，所述制剂的膜材中，仅含有磷脂，或者包括磷脂和阳离子脂质，或者包括磷脂和靶向分子，或者包括磷脂、阳离子脂质和靶向分子。In the present invention, the membrane material of the preparation contains only phospholipids, or includes phospholipids and cationic lipids, or includes phospholipids and targeting molecules, or includes phospholipids, cationic lipids and targeting molecules.
其中：in:
磷脂与阳离子脂质的质量比为(0.1～10):1，一些实施例中，磷脂与阳离子脂质的质量比为(1～10):1，一些实施例中，磷脂与阳离子脂质的质量比为(0.1～1):1，一些实施例中，磷脂与阳离子脂质的质量比为(0.5～5):1。一些具体实施例中，磷脂与阳离子脂质的质量比为0.5:1、0.6:1、0.7:1、0.8:1、0.9:1、1:1、2:1、3:1、4:1或5:1。The mass ratio of phospholipid to cationic lipid is (0.1-10):1. In some embodiments, the mass ratio of phospholipid to cationic lipid is (1-10):1. In some embodiments, the mass ratio of phospholipid to cationic lipid is (0.1-1):1. In some embodiments, the mass ratio of phospholipid to cationic lipid is (0.5-5):1. In some specific embodiments, the mass ratio of phospholipid to cationic lipid is 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 2:1, 3:1, 4:1 or 5:1.
磷脂与靶向分子的质量比为(10～100):(2～20)。一些实施例中，磷脂与靶向分子的质量比为(10～25):(2～10)。更具体的，磷脂与靶向分子的质量比为(15～20):(2～5)。作为优选，磷脂与靶向分子的质量比为15:(2～5)，或为16:(2～5)，或为17:(2～5)，或为18:(2～5)，或为19:(2～5)，或为20:(2～5)，更优选的，磷脂与靶向分子的质量比为(15～16):2、或为(15～16):3、或为(15～16):4、或为(15～16):5；或为(16～17):2、或为(16～17):3、或为(16～17):4、或为(16～17):5；或为(17～18):2、或为(17～18):3、或为(17～18):4、或为(17～18):5；或为(18～19):2、或为(18～19):3、或为(18～19):4、或为(18～19):5；或为(19～20):2、或为(19～20):3、或为(19～20):4、或为(19～20):5。The mass ratio of phospholipids to targeting molecules is (10-100):(2-20). In some embodiments, the mass ratio of phospholipids to targeting molecules is (10-25):(2-10). More specifically, the mass ratio of phospholipids to targeting molecules is (15-20):(2-5). Preferably, the mass ratio of phospholipids to targeting molecules is 15:(2-5), or 16:(2-5), or 17:(2-5), or 18:(2-5), or 19:(2-5), or 20:(2-5). More preferably, the mass ratio of phospholipids to targeting molecules is (15-16):2, or (15-16):3, or (15-16):4, or (15-16):5; or (16-17):2, or (16-17):3, Or it is (16-17):4, or it is (16-17):5; or it is (17-18):2, or it is (17-18):3, or it is (17-18):4, or it is (17-18):5; or it is (18-19):2, or it is (18-19):3, or it is (18-19):4, or it is (18-19):5; or it is (19-20):2, or it is (19-20):3, or it is (19-20):4, or it is (19-20):5.
磷脂、阳离子脂质和靶向分子的质量比为(1～10):(1-10):(0.2-2)The mass ratio of phospholipid, cationic lipid and targeting molecule is (1-10):(1-10):(0.2-2)
一些实施例中，所述新藤黄酸、磷脂和中性脂的质量比为(0.01～0.2):(0.5～4):(2～10)；具体的，所述新藤黄酸、磷脂和中性脂的质量比为(0.01～0.1):(0.5～4):(2～10)，或者所述新藤黄酸、磷脂和中性脂的质量比为(0.01～0.2):(1～4):(2～10)，或者所述新藤黄酸、磷脂和中性脂的质量比为(0.01～0.2):(0.5～4):(2～7)，更具体的，所述新藤黄酸、磷脂和中性脂的质量比为(0.01～0.1):(1～4):(2～7)，作为优选，所述新藤黄酸、磷脂和中性脂的质量比为(0.05～0.1):(1～3):(4～6)，更优选的，所述新藤黄酸、磷脂和中性脂的质量比为(0.05～0.1):2:5，或为0.09:(1～3):5，或为0.09:2:(4～6)。在一些具体实施例中，磷脂为蛋黄卵磷脂，中性脂为三油酸甘油酯，则所述新藤黄酸、蛋黄卵磷脂和三油酸甘油酯的质量比为0.09:2:5。In some embodiments, the mass ratio of the neogamoic acid, phospholipids and neutral lipids is (0.01-0.2):(0.5-4):(2-10); specifically, the mass ratio of the neogamoic acid, phospholipids and neutral lipids is (0.01-0.1):(0.5-4):(2-10), or the mass ratio of the neogamoic acid, phospholipids and neutral lipids is (0.01-0.2):(1-4):(2-10), or the mass ratio of the neogamoic acid, phospholipids and neutral lipids is (0.01-0.2): (0.5-4): (2-7), more specifically, the mass ratio of the neogamoic acid, phospholipids and neutral lipids is (0.01-0.1): (1-4): (2-7), preferably, the mass ratio of the neogamoic acid, phospholipids and neutral lipids is (0.05-0.1): (1-3): (4-6), more preferably, the mass ratio of the neogamoic acid, phospholipids and neutral lipids is (0.05-0.1): 2: 5, or 0.09: (1-3): 5, or 0.09: 2: (4-6). In some specific embodiments, the phospholipid is egg yolk lecithin and the neutral lipid is triolein, then the mass ratio of the neogamoic acid, egg yolk lecithin and triolein is 0.09: 2: 5.
一些实施例中，所述新藤黄酸、磷脂、中性脂和阳离子脂质的质量比为(0.01～0.2):(0.5～4):(2～10):(0.1～4)。具体的，所述新藤黄酸、磷脂、中性脂和阳离子脂质的质量比为(0.01～0.1):(0.5～4):(2～10):(0.1～4)，或者所述新藤黄酸、磷脂、中性脂和阳离子脂质的质量比为(0.01～0.2):(1～4):(2～10):(0.1～4)，或者所述新藤黄酸、磷脂、中性脂和阳离子脂质的质量比为(0.01～0.2):(0.5～4):(2～7):(0.1～4)，或者所述新藤黄酸、磷脂、中性脂和阳离子脂质的质量比为(0.01～0.2):(0.5～4):(2～10):(0.1～1)；更具体的，所述新藤黄酸、磷脂、中性脂和阳离子脂质的质量比为(0.01～0.1):(1～4):(2～7):(0.1～1)，作为优选，所述新藤黄酸、磷脂、中性脂和阳离子脂质的质量比为(0.05～0.1):(1～3):(4～6):(0.1～1)，更优选的，所述新藤黄酸、磷脂、中性脂和阳离子脂质的质量比为(0.05～0.1):1.5:5:0.5，或为0.09:(1～3):5:0.5，或为0.09:2:(4～6):0.5，或为0.09:2:1.5:(0.1～1)。在一些具体实施例中，磷脂为蛋黄卵磷脂，中性脂为三油酸甘油酯，阳离子脂质为DOTAP，则所述新藤黄酸、蛋黄卵磷脂、三油酸甘油酯和DOTAP的质量比为0.09:1.5:5:0.5。In some embodiments, the mass ratio of the neogamoic acid, phospholipids, neutral lipids and cationic lipids is (0.01-0.2): (0.5-4): (2-10): (0.1-4). Specifically, the mass ratio of the neogamoic acid, phospholipids, neutral lipids and cationic lipids is (0.01-0.1): (0.5-4): (2-10): (0.1-4), or the mass ratio of the neogamoic acid, phospholipids, neutral lipids and cationic lipids is (0.01-0.2): (1-4): (2-10): (0.1-4), or the mass ratio of the neogamoic acid, phospholipids, neutral lipids and cationic lipids is (0.01-0.2): (1-4): (2-10): (0.1-4). 1～0.2):(0.5～4):(2～7):(0.1～4), or the mass ratio of the new gambogic acid, phospholipids, neutral lipids and cationic lipids is (0.01～0.2):(0.5～4):(2～10):(0.1～1); More specifically, the mass ratio of the new gambogic acid, phospholipids, neutral lipids and cationic lipids is (0.01～0.1):(1～4):(2～7):(0.1～1), Preferably, the mass ratio of the neogamoic acid, phospholipid, neutral lipid and cationic lipid is (0.05-0.1):(1-3):(4-6):(0.1-1), more preferably, the mass ratio of the neogamoic acid, phospholipid, neutral lipid and cationic lipid is (0.05-0.1):1.5:5:0.5, or 0.09:(1-3):5:0.5, or 0.09:2:(4-6):0.5, or 0.09:2:1.5:(0.1-1). In some specific embodiments, the phospholipid is egg yolk lecithin, the neutral lipid is triolein, and the cationic lipid is DOTAP, then the mass ratio of the neogamoic acid, egg yolk lecithin, triolein and DOTAP is 0.09:1.5:5:0.5.
相对于其他案例，磷脂为蛋黄卵磷脂，中性脂为三油酸甘油酯，阳离子脂质为DOTAP，且所述新藤黄酸、蛋黄卵磷脂、三油酸甘油酯和DOTAP的质量比为0.09:1.5:5:0.5，能够获得更好的包封效果，生物利用度更好，且能够进一步提高抗肿瘤效果。Compared with other cases, the phospholipid is egg yolk lecithin, the neutral lipid is triolein, the cationic lipid is DOTAP, and the mass ratio of the new gamboge acid, egg yolk lecithin, triolein and DOTAP is 0.09:1.5:5:0.5, which can obtain better encapsulation effect, better bioavailability, and can further improve the anti-tumor effect.
进一步的，本发明还提供了如前所述制剂的制备方法，包括：Furthermore, the present invention also provides a method for preparing the above-mentioned preparation, comprising:
将新藤黄酸溶液和中性脂混合后，与缓冲液和单分子磷脂膜的膜材混合，经反复涡旋、离心后得到包载新藤黄酸的脂肪体；The neogambogic acid solution and neutral lipid are mixed, and then mixed with a buffer solution and a monolayer phospholipid membrane material, and the neogambogic acid-encapsulated fat body is obtained after repeated vortexing and centrifugation;
所述膜材包括磷脂或功能性极性脂质中的一种或多种。一些实施例中，所述膜材中还包括阳离子脂质和/或靶向分子。The membrane material comprises one or more of phospholipids or functional polar lipids. In some embodiments, the membrane material further comprises cationic lipids and/or targeting molecules.
即，本发明所述的膜材中包括磷脂，或者包括磷脂和阳离子脂质，或者包括磷脂和靶向分子，或者包括磷脂、阳离子脂质和靶向分子。That is, the membrane material of the present invention includes phospholipids, or includes phospholipids and cationic lipids, or includes phospholipids and targeting molecules, or includes phospholipids, cationic lipids and targeting molecules.
所述缓冲液为PBS缓冲液、HEPES缓冲液、蔗糖溶液、NaCl溶液、KCl溶液或MgCl2溶液。The buffer solution is PBS buffer solution, HEPES buffer solution, sucrose solution, NaCl solution, KCl solution orMgCl2 solution.
所述膜材中，不仅仅含有磷脂，则膜材中各组分分别以有机溶剂溶解后混合，再挥去有机溶剂。所述有机溶剂为甲醇与如下溶剂中至少一种的混合物：所述溶剂包括无水乙醇、氯仿、苯、甲苯、二甲苯、丁醇、异丙醇、乙醚、丙酮、环己酮、甲基异丁基酮、醋酸乙酯、醋酸丁酯、环己酮或石油醚中，优选为甲醇和氯仿混合溶液；The membrane material contains more than just phospholipids, and the various components of the membrane material are dissolved in an organic solvent, mixed, and then the organic solvent is evaporated. The organic solvent is a mixture of methanol and at least one of the following solvents: anhydrous ethanol, chloroform, benzene, toluene, xylene, butanol, isopropanol, ethyl ether, acetone, cyclohexanone, methyl isobutyl ketone, ethyl acetate, butyl acetate, cyclohexanone, or petroleum ether, preferably a mixture of methanol and chloroform.
如前所述方法中，所述反复涡旋、离心包括：混合后，经涡旋得到混合物1，所述混合物1经离心收集下层溶液后，再次涡旋得到混合物2；所述混合物2经离心去除沉淀后，再次涡旋得到混合物3；所述混合物3经离心收集下层溶液，再次涡旋得到含有所述脂肪体的混合物4。As described above, the repeated vortexing and centrifugation include: after mixing, vortexing to obtain mixture 1, centrifuging the mixture 1 to collect the lower layer solution, and vortexing again to obtain mixture 2; centrifuging the mixture 2 to remove the precipitate, and vortexing again to obtain mixture 3; centrifuging the mixture 3 to collect the lower layer solution, and vortexing again to obtain mixture 4 containing the fat body.
具体的：Specifically:
混合后，经涡旋得到混合物1。After mixing, the mixture was vortexed to obtain Mixture 1.
在该步骤中，所述涡旋的参数包括:3000-4000rpm涡旋3～7min，工作1～10s，停止1～10s。作为优选，涡旋的转速为3000、3200、3400、3500、3600、3700、3800、3900或4000rpm。作为优选，所述涡旋的时长为3min、4min、5min、6min或7min。作为优选，所述涡旋每工作1s停止1s，或每工作2s停止2s，或每工作2s停止2s，或每工作3s停止3s，或每工作4s停止4s，或每工作5s停止5s，或每工作6s停止6s，或每工作7s停止7s，或每工作8s停止8s，或每工作9s停止9s，或每工作10s停止10s，或每工作5s停止6s，或每工作5s停止7s，或每工作5s停止8s，或每工作5s停止9s，或每工作5s停止10s，或每工作6s停止7s，或每工作6s停止8s，或每工作6s停止9s，或每工作6s停止10s，或每工作7s停止8s，或每工作7s停止9s，或每工作7s停止10s，或每工作8s停止9s，或每工作8s停止10s，或每工作9s停止10s。一些具体实施例中，该步骤中所述涡旋的参数包括4000rpm，涡旋10s停止10s，涡旋4min。或者该步骤中的涡旋参数包括4000rpm，涡旋10s停止5s，涡旋3min。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.
所述混合物1经离心收集下层溶液后，再次涡旋得到混合物2。The mixture 1 was centrifuged to collect the lower layer solution, and then vortexed again to obtain a mixture 2.
在该步骤中，所述涡旋的参数包括1000-4000rpm，所述离心的参数包括：常温800-1200×g离心3-7min。作为优选，所述离心的转速为800×g、900×g、1000×g、1100×g或1200×g，所述离心的时长为3min、4min、5min、6min或7min。一些实施例中，所述离心条件包括常温1000×g离心5min。In this step, the vortexing parameters include 1000-4000 rpm, and the centrifugation parameters include: centrifugation at 800-1200×g for 3-7 minutes 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 minutes, 4 minutes, 5 minutes, 6 minutes, or 7 minutes. In some embodiments, the centrifugation conditions include centrifugation at 1000×g for 5 minutes at room temperature.
所述混合物2经离心去除沉淀后，再次涡旋得到混合物3；After the mixture 2 is centrifuged to remove the precipitate, it is vortexed again to obtain the mixture 3;
在该步骤中，所述涡旋的参数包括1000-4000rpm，所述离心的参数包括：实际应用中18000-22000×g离心3-7min。作为优选，所述离心的转速为18000×g、19000×g、20000×g、21000×g或22000×g，所述离心的时长为3min、4min、5min、6min或7min。一些实施例中，所述离心条件包括常温20000×g离心5min。In this step, the vortexing parameters include 1000-4000 rpm, and the centrifugation parameters include: in actual applications, centrifugation at 18,000-22,000 × g for 3-7 minutes. Preferably, the centrifugation speed is 18,000 × g, 19,000 × g, 20,000 × g, 21,000 × g, or 22,000 × g, 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 20,000 × g for 5 minutes at room temperature.
所述混合物3经离心收集下层溶液，再次涡旋得到含有所述脂肪体的混合物4。在该步骤中，所述涡旋的参数包括1000-4000rpm，所述离心的参数包括：实际应用中800-1200×g离心3-7min。作为优选，所述离心的转速为800×g、900×g、1000×g、1100×g或1200×g，所述离心的时长为3min、4min、5min、6min或7min。一些实施例中，所述离心条件包括常温1000×g离心5min。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 1000-4000rpm, and the centrifugal parameters include: in actual application, 800-1200×g centrifugation for 3-7min. As an example, the centrifugal The rotation 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 condition includes centrifugation at 1000×g for 5 min at room temperature.
本发明提供的制备方法工艺简单，容易操作，获得的制剂具有良好的载药量和生物利用度。经验证，该方法制备的新藤黄酸脂肪体制剂中新藤黄酸的含量到达1mg/ml，浓度是其在水中的2000倍。同时，新藤黄酸脂肪体具有良好的抗肿瘤效果，可以抑制结直肠癌、乳腺癌、肝癌等多种癌细胞的生长，效果显著优于游离的新藤黄酸。The preparation method provided by the present invention is simple and easy to operate, and the obtained preparation has good drug loading and bioavailability. It has been verified that the content of neogambogic acid in the neogambogic acid fat body preparation prepared by this method reaches 1 mg/ml, which is 2000 times the concentration in water. At the same time, the neogambogic acid fat body has a good anti-tumor effect, which can inhibit the growth of various cancer cells such as colorectal cancer, breast cancer, and liver cancer, and the effect is significantly better than that of free neogambogic acid.
进一步的，本发明提供了如前所述制剂或如前所述方法制备获得的制剂，在制备药物或疫苗中的应用。Furthermore, the present invention provides the use of the above-mentioned preparation or the preparation obtained by the above-mentioned method in the preparation of medicines or vaccines.
所述药物和/或疫苗的功效包括：抗氧化、抗炎、抗癌和抗菌中至少一种。The efficacy of the drug and/or vaccine includes at least one of anti-oxidation, anti-inflammatory, anti-cancer and antibacterial.
一些实施例中，所述癌包括：乳腺癌、肺癌、前列腺癌、胃癌、头颈癌、肾癌、喉癌、肝癌、肌肉组织癌症、血液瘤、骨癌、脑癌、口腔或鼻黏膜癌、膀胱癌、中枢神经系统癌症、宫颈癌、头颈癌、结肠癌、子宫内膜癌、外生殖器癌、食管癌、胆囊癌、胃肠道癌、泌尿生殖道癌、卵巢癌、胰腺癌、皮肤癌、脾癌、小肠癌、大肠癌、黑色素瘤、睾丸癌和/或甲状腺癌。In some embodiments, the cancer comprises breast cancer, lung cancer, prostate cancer, stomach cancer, head and neck cancer, kidney cancer, laryngeal cancer, liver cancer, muscle tissue cancer, blood tumor, bone cancer, brain 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, ovarian cancer, pancreatic cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, melanoma, testicular cancer and/or thyroid cancer.
更进一步的，本发明提供了一种药物或疫苗，其包括如前所述制剂或所述方法制备获得的制剂。Furthermore, the present invention provides a medicine or vaccine, which includes the preparation as described above or the preparation prepared by the method.
本发明所述的药物或疫苗中还包括有效量的肿瘤抑制剂；The medicine or vaccine of the present invention further comprises an effective amount of a tumor inhibitor;
所述肿瘤抑制剂包括：顺铂、卡铂、奥沙利铂、5-氟脲嘧啶(5-FU)、甲氨蝶呤、柔红霉素、放线菌素-D、伊立替康(CPT-11)、米托蒽醌、雌莫司汀、长春新碱、地塞米松、强的松、洛莫司汀、甲氨蝶呤、吡柔比星、阿霉素、吉西他滨、奎扎替尼或贝伐单抗。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.
本发明所述的药物或疫苗中，还包括药学上可接受的辅料。The medicine or vaccine of the present invention further includes pharmaceutically acceptable excipients.
本发明中，所述药学上可接受的辅料，根据剂型的需要进行选择。In the present invention, the pharmaceutically acceptable excipients are selected according to the requirements of the dosage form.
本发明所述的药物或疫苗的剂型为口服制剂、吸入剂或注射剂。The dosage form of the medicine or vaccine of the present invention is oral preparation, inhalant or injection.
可选择的，所述药物或疫苗的为口服制剂，例如，其可为片剂、丸剂、口服液剂、胶囊剂、糖浆剂、滴丸剂或颗粒剂。Alternatively, the drug or vaccine is in the form of an oral preparation, for example, a tablet, a pill, Oral solution, capsule, syrup, pill or granule.
在本发明提供的一些实施例中，胶囊剂为硬胶囊剂或软胶囊剂。In some embodiments provided herein, the capsule is a hard capsule or a soft capsule.
在本发明提供的一些实施例中，片剂为口服片剂或口腔片剂。In some embodiments provided herein, the tablet is an oral tablet or buccal tablet.
口服片剂指供口服的片剂，多数此类片剂中的药物是经胃肠道吸收而发挥作用，也有的片剂中的药物是在胃肠道局部发挥作用。在本发明提供的一些实施例中，口服片剂为普通压制片、分散片、泡腾片、咀嚼片、包衣片或缓控释片。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.
所述药物或疫苗为吸入剂，可选择的，其为吸入气雾剂、吸入粉雾剂和供雾化器使用的液体制剂。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.
所述药物或疫苗为注射剂，例如，其为注射液剂或注射用粉针剂。The medicine or vaccine is an injection, for example, an injection solution or an injection powder.
本发明还提供了一种防治肿瘤的方法，其包括给予如前所述的药物或疫苗。所述给予的方式包括口服、吸入和/或注射。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.
所述方法的受试者为人类或者，所述方法的受试者为灵长类动物或非灵长类哺乳动物。The subject of the method is a human or, the subject of the method is a primate or a non-primate mammal.
本发明提供的新型纳米颗粒脂肪体(具有一个疏水的核心)包载新藤黄酸构建新藤黄酸脂肪体，并通过不同的磷脂来修饰新藤黄酸脂肪体增加其有效性或安全性。同时本发明还提供了其制备方法，制备的新藤黄酸脂肪体制剂中新藤黄酸的含量到达1mg/ml，浓度是其在水中的2000倍。显著提高其生物利用度和适用范围。同时，新藤黄酸脂肪体具有良好的抗肿瘤效果，可以抑制结直肠癌、乳腺癌、肝癌等多种癌细胞的生长，效果显著优于游离的新藤黄酸。The novel nanoparticle fat body (having a hydrophobic core) provided by the present invention encapsulates neogamonic acid to construct a neogamonic acid fat body, and the neogamonic acid fat body is modified by different phospholipids to increase its effectiveness or safety. At the same time, the present invention also provides a preparation method thereof. The content of neogamonic acid in the prepared neogamonic acid fat body preparation reaches 1 mg/ml, which is 2000 times its concentration in water. Its bioavailability and scope of application are significantly improved. At the same time, the neogamonic acid fat body has a good anti-tumor effect and can inhibit the growth of various cancer cells such as colorectal cancer, breast cancer, and liver cancer, and the effect is significantly better than that of free neogamonic acid.
图1示包载新藤黄酸构建新藤黄酸脂肪体；Figure 1 shows the encapsulation of neo-gambogic acid to construct neo-gambogic acid fat bodies;
图2示制备新藤黄酸脂肪体，其中：A，不带NGA的脂肪体(空载脂肪体，即Blank-adiposome,Blank-AD)的光学显微镜照片(a-c)和电镜照片(d)；B，带有NGA但不带阳离子的脂肪体(新藤黄酸脂肪体，即NGA-adiposome without cationic,NGA-AD)的光学显微镜照片(a-c)和电镜照片(d)；C，带有NGA的带阳离子的脂肪体(新藤黄酸阳离子脂肪体，即NGA-adiposome with cationic,NGA-C-AD)的光学显微镜照片(a-c)和电镜照片(d)；a是DIC的结果，b是中性脂染料Nile Red的染色结果，c是图a和图b融合的结果，图d是电子显微镜结果；图a-c中的比例尺是5微米，d中的比例尺是10nm；D，动态光散射仪检测了这些脂肪体的平均粒径和PDI(聚合物分散性指数)；E，TLC检测这些脂肪体中的脂质组成和NGA的信号；泳道1-3是标准品，分别为Egg PC，DOTAP，NGA；泳道4-6分别是Blank-AD，NGA-AD，和NGA-C-AD；F，HPLC检测乙醇中游离的NGA标准品和新藤黄酸脂肪体中的NGA；G，制备的新藤黄酸脂肪体表面的Ztea电位；Figure 2 shows the preparation of new gambogic acid fat bodies, wherein: A, optical microscope photos (ac) and electron microscope photos (d) of fat bodies without NGA (empty fat body, i.e., Blank-AD); B, optical microscope photos (ac) and electron microscope photos (d) of fat bodies with NGA but without cations (new gambogic acid fat body, i.e., NGA-adiposome without cationic, NGA-AD); C, cationic fat bodies with NGA (new gambogic acid cationic Optical micrographs (ac) and electron micrographs (d) of fat bodies, i.e., NGA-adiposome with cationic, NGA-C-AD); a is the result of DIC, b is the result of staining with the neutral lipid dye Nile Red, c is the result of merging Figures a and b, and Figure d is the result of electron microscopy; the scale bars in Figures ac and d are 5 μm, and the scale bar in d is 10 nm; D, dynamic light scattering instrument detected the average particle size and PDI (polymer dispersibility index) of these fat bodies; E, TLC detected the lipid composition and NGA signal in these fat bodies; lanes 1-3 are standards, namely Egg PC, DOTAP, and NGA; lanes 4-6 are Blank-AD, NGA-AD, and NGA-C-AD, respectively; F, HPLC detection of free NGA standards in ethanol and NGA in new gambogic acid fat bodies; G, Ztea potential on the surface of prepared new gambogic acid fat bodies;
图3示新藤黄酸脂肪体具有抗肿瘤活性，效果显著优于游离的新藤黄酸实施例1中制备的不同的新藤黄酸脂肪体分别按照图示的浓度处理相对应的细胞，并通过CCK8测量细胞的存活率；A，直肠癌细胞CT26；B，乳腺癌细胞4T1；C，人血液瘤细胞H929；D，人血液瘤细胞Raji；其中，ns表示无显著性,*p<0.05,**p<0.01,***p<0.001；Figure 3 shows that the new gambogic acid fat body has anti-tumor activity, and the effect is significantly better than that of free new gambogic acid. The different new gambogic acid fat bodies prepared in Example 1 were treated with the corresponding cells according to the concentrations shown, and the cell survival rate was measured by CCK8; A, rectal cancer cell CT26; B, breast cancer cell 4T1; C, human hematologic malignant tumor cell H929; D, human hematologic malignant tumor cell Raji; where ns indicates not significant, *p<0.05, **p<0.01, ***p<0.001;
图4示新藤黄酸脂肪体抑制小鼠结直肠癌的生长，效果优于游离的新藤黄酸，其中：A，不同处理组小鼠肿瘤的生长曲线变化；B，不同处理组的小鼠体重变化；C，不同处理组小鼠肿瘤的照片；D，不同处理组小鼠肿瘤的重量和肿瘤抑制率；E，不同处理组小鼠肿瘤的安全性评价；RBC，红细胞数量；HGB，血常规指标血红蛋白；Cre，肾脏炎症指标肌酐；AST，肝脏炎症指标天冬氨酸转氨酶；Figure 4 shows that neogambogic acid fat bodies inhibit the growth of colorectal cancer in mice, and the effect is better than that of free neogambogic acid, where: A, growth curve of tumors in mice in different treatment groups; B, weight changes of mice in different treatment groups; C, photos of tumors in mice in different treatment groups; D, weight and tumor inhibition rate of tumors in mice in different treatment groups; E, safety evaluation of tumors in mice in different treatment groups; RBC, red blood cell count; HGB, hemoglobin, a blood test indicator; Cre, creatinine, an indicator of kidney inflammation; AST, aspartate aminotransferase, an indicator of liver inflammation;
图5示新藤黄酸脂肪体抑制小鼠肝癌的生长，效果优于游离的新藤黄酸，其中：A，不同处理组小鼠肝脏肿瘤的照片；B，不同处理组小鼠肝脏肿瘤的重量和肿瘤抑制率；C，不同处理组的小鼠体重变化；D，不同处理组小鼠肿瘤的安全性评价；RBC，红细胞数量；HGB，血常规指标血红蛋白；Cre，肾脏炎症指标肌酐；AST，肝脏炎症指标天冬氨酸转氨酶。Figure 5 shows that the neogamoic acid fat body inhibits the growth of liver cancer in mice, and its effect is better than that of free neogamoic acid, among which: A, photos of liver tumors in mice in different treatment groups; B, weights and tumor inhibition rates of liver tumors in mice in different treatment groups; C, weight changes of mice in different treatment groups; D, safety evaluation of tumors in mice in different treatment groups; RBC, red blood cell count; HGB, hemoglobin, a blood routine indicator; Cre, creatinine, an indicator of kidney inflammation; AST, aspartate aminotransferase, an indicator of liver inflammation.
本发明提供了新藤黄酸脂肪体制剂及其制备方法和应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。The present invention provides a new gambogic acid fat body preparation and its preparation method and application. Personnel can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out 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 personnel 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.
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by one of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). Amino acid residue abbreviations are the standard three-letter and/or one-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
本申请中，术语“和/或”，描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示:单独存在A，同时存在A和B，单独存在B的情况。其中A,B可以是单数或者复数。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.
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。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.
本申请中，“至少一个”是指一个或者多个，“多个”是指两个或两个以上。“以下至少一项(个)”或其类似表达，是指的这些项中的任意组合，包括单项(个)或复数项(个)的任意组合。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.
本文术语“药物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。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.
本文术语“防治”包括预防和/或治疗。所述“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症和肿瘤。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。The term "prevention" herein includes prevention and/or treatment. The "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the subject, such as cancer and tumors. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of disease severity, 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 or complete), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease as well as subjects susceptible to a condition or disease. The subject or subject for whom the condition or disease is to be prevented. When terms such as alleviate, lessen, attenuate, moderate, or alleviate are used, they also include elimination, disappearance, and non-occurrence.
本文术语“给予”的对象是指接受对如本发明所述的特定疾病或病症的治疗的生物体。示例性地，接受疾病或病症治疗的是哺乳动物，如人、灵长类动物(例如，猴)或非灵长类哺乳动物。The subject of the term "administered" herein refers to an organism that receives treatment for a specific 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 (e.g., a monkey), or a non-primate mammal.
本文术语“受试者”是指接受对如本发明所述的特定疾病或病症的治疗的生物体。示例性地，“受试者”包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵长类哺乳动物。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.
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。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.
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。本文术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。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.
本文术语“IC50”是指被测量的拮抗剂的半抑制浓度。可以理解为一定浓度的某种药物诱导肿瘤细胞死亡50％，该浓度称为50％抑制浓度，即死亡细胞与全部细胞数之比等于50％时所对应的浓度，IC50值可以用来衡量药物诱导死亡的能力，即诱导能力越强，该数值越低，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.
应理解，在本申请的各种实施例中，上述各过程的序号的大小并不意味着执行顺序的先后，部分或全部步骤可以并行执行或先后执行，各过程的执行顺序应以其功能和内在逻辑确定，而不应对本申请实施例的实施过程构成任何限定。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.
本发明采用的试材皆为普通市售品，皆可于市场购得。The test materials used in the present invention are all common commercial products and can be purchased in the market.
本文中涉及片段名称及序列如下：The fragment names and sequences involved in this article are as follows:
表1靶向识别单分子磷脂膜的肽段的名称和氨基酸序列
Table 1 Names and amino acid sequences of peptides targeting single-molecule phospholipid membranes
表2靶向分子名称和氨基酸序列及靶向蛋白
Table 2 Targeting molecule names and amino acid sequences and targeting proteins
本发明用纳米颗粒脂肪体包载新藤黄酸，构建新藤黄酸醇脂肪体。制备的新藤黄酸脂肪体制剂中新藤黄酸的含量到达1mg/ml，浓度是其在水中的2000倍。同时，新藤黄酸脂肪体具有良好的抗肿瘤效果，可以抑制结直肠癌、乳腺癌、肝癌等多种癌细胞的生长，效果显著优于游离的新藤黄酸。因此，本发明提供了一种新藤黄酸脂肪体和其制备方法，从而显著提高了新藤黄酸的溶解度，并有效地抑制肿瘤的生长，扩大了新藤黄酸的应用范围，为新藤黄酸的临床转化奠定了基础，在癌症治疗中具有广阔和富有前景的应用市场。下面结合实施例，进一步阐述本发明。The present invention uses nanoparticle fat bodies to encapsulate neogamoic acid to construct neogamoic acid alcohol fat bodies. The content of neogamoic acid in the prepared neogamoic acid fat body preparation reaches 1 mg/ml, and its concentration is 2000 times that in water. At the same time, the neogamoic acid fat body has a good anti-tumor effect and can inhibit the growth of various cancer cells such as colorectal cancer, breast cancer, and liver cancer. The effect is significantly better than that of free neogamoic acid. Therefore, the present invention provides a neogamoic acid fat body and a preparation method thereof, thereby significantly improving the solubility of neogamoic acid, effectively inhibiting the growth of tumors, and expanding the application range of neogamoic acid. The scope of application has laid a foundation for the clinical transformation of new gambogic acid, and has a broad and promising application market in cancer treatment. The present invention is further described below in conjunction with the examples.
实施例1制备新藤黄酸脂肪体(NGA-adiposome,NGA-AD)Example 1 Preparation of New Gambogic Acid Adiposome (NGA-AD)
1)中性脂：三油酸甘油酯(triolein)。1) Neutral fat: triolein.
2)用三油酸甘油酯直接溶解NGA至饱和。用高效液相色谱(HPLC)检测三油酸甘油酯中的NGA含量为18.3mg/ml。HPLC的测条件：色谱柱为Agilent Zorbax SB-C18，流动相为甲醇：乙腈：水＝6:3:1(v/v/v，0.1％三氯乙酸，m％)，柱温35℃，检测波长360nm，流速1ml/min。2) NGA was directly dissolved in triolein to saturation. High-performance liquid chromatography (HPLC) determined the NGA content in triolein to be 18.3 mg/ml. HPLC conditions: Agilent Zorbax SB-C18 column, mobile phase of methanol:acetonitrile:water = 6:3:1 (v/v/v, 0.1% trichloroacetic acid, m%), column temperature 35°C, detection wavelength 360 nm, flow rate 1 ml/min.
3)磷脂：氯仿分别配制蛋黄卵磷脂(EggPC)和阳离子脂质(2,3-二油酰基-丙基)-三甲胺(氯盐)(DOTAP)浓度为25mg/ml。3) Phospholipids: Egg yolk phosphatidylcholine (EggPC) and cationic lipid (2,3-dioleoyl-propyl)-trimethylamine (chloride) (DOTAP) were prepared in chloroform at a concentration of 25 mg/ml.
4)取60μl上述配制的Egg PC和20μl上述配制的DOTAP加入到微量离心管中，用高纯氮气吹干溶剂。4) Take 60μl of the above-prepared Egg PC and 20μl of the above-prepared DOTAP and add them to a microcentrifuge tube, and blow dry the solvent with high-purity nitrogen gas.
5)向微量离心管中加入100μl PBS和5μl步骤2)制备的得到含有NGA的中性脂，涡旋4min(涡旋10s，停止10s)(实际应用中涡旋3-7min均可，涡旋条件为3000-4000rpm，本实施例用的是4000rpm)，得到乳白色的脂质混合物1，将该脂质混合物1以1000×g离心5min(实际应用中800-1200×g离心3-7min均可)，离心后，液相体系呈现两层分层，通过抽取的方式收集下层乳白色溶液，涡旋，得到乳白色的脂质混合物2。5) Add 100 μl PBS and 5 μl neutral lipid containing NGA prepared in step 2) into a microcentrifuge tube, vortex for 4 min (vortex for 10 s, stop for 10 s) (in actual application, vortex for 3-7 min, vortex conditions are 3000-4000 rpm, and 4000 rpm is used in this embodiment) to obtain a milky white lipid mixture 1, and centrifuge the lipid mixture 1 at 1000 × g for 5 min (in actual application, centrifuge at 800-1200 × g for 3-7 min). After centrifugation, the liquid phase system presents two layers. Collect the lower milky white solution by extraction and vortex to obtain a milky white lipid mixture 2.
6)步骤5)得到的脂质混合物2以20000×g离心5min(实际应用中18000-22000×g离心3-7min均可)。离心后，去除微量离心管中底部的沉淀组分，涡旋，得到乳白色的脂质混合物3。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.
7)将步骤6)得到的脂质混合物3以1000×g离心5min(实际应用中800-1200×g离心3-7min均可)，离心后，液相体系呈现两层分层，通过抽取的方式收集下层乳白色溶液，涡旋，得到乳白色的脂质混合物4，即为最终的携带疏水性小分子化合物新藤黄酸的带阳离子的脂肪体(新藤黄酸阳离子脂肪体，即NGA-adiposome with cationic,NGA-C-AD)。同样的制作方法，我们制作了带有NGA但不带阳离子的脂肪体(新藤黄酸脂肪体，即NGA-adiposome without cationic,NGA-AD)。同样的制作方法，我们制作了不带NGA的脂肪体(空载脂肪体，即Blank-adiposome,Blank-AD)。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 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 cationic fat body carrying the hydrophobic small molecule compound neogamonic acid (neogamonic acid cationic fat body, i.e., NGA-adiposome with cationic, NGA-C-AD). Using the same preparation method, we prepared fat bodies with NGA but without cations (neogamonic acid fat body). We also prepared liposomes without NGA (blank-adiposome, Blank-AD) using the same method.
通过光学显微镜和电子显微镜观察构建的上述三种脂肪体的形态结构，它们都是均一的球状结构，并且没有其它膜杂质的污染(图2中A,B和C)。另外，通过动态光散射仪检测了这些脂肪体的平均粒径为120nm，PDI(分散性指数)为0.15(图2中D)。进一步通过分别通过TLC(薄层色谱法)和HPLC(高效液相色谱法)检测新藤黄酸脂肪体中是否含有新藤黄酸。TLC检测方法为：将上述构建好的新藤黄酸脂肪体加入相同体积的甲醇和2倍体积的氯仿萃取脂质，收集有机相，用氮气吹干，得到总脂质。在获得的总脂质加入100μl氯仿中，上样10μl至硅胶板，在正己烷：乙醚：冰乙酸(体积比为80:20:1)的展开剂中展开，以分离三油酸甘油酯。然后再将硅胶板放置在氯仿：甲醇：冰乙酸：水(体积比为75:13:9:3)的溶剂中展开，以分离EggPC,DOTAP和NGA。TLC检测结果中根据标准品的位置判断脂肪体中是否有NGA的信号，在泳道4(Blank-AD)中没有检测到NGA信号，在泳道5(NGA-AD)中检测到NGA的信号，但是没有DOTAP的信号，在泳道6(NGA-C-AD)中检测到NGA的信号和DOTAP的信号。但是这些脂肪体中都有三油酸甘油酯的信号(图2中E)。在HPLC结果中，新藤黄酸脂肪体中NGA的出峰时间与乙醇中游离的NGA标准品的出峰时间一致(图2中F)。同时，Ztea电位结果显示，NGA-C-AD带有正电为60mV左右，而Blank-AD和NGA-AD则不带正电，分别为0mV和-20mV(图2中G)以上结果表明，成功地制备了含有新藤黄酸的脂肪体，并且其纯度高，均一性好。The morphological structures of the three constructed fat bodies were observed using optical and electron microscopy, revealing uniform spherical structures free of contamination from other membrane impurities (Figure 2, A, B, and C). Furthermore, dynamic light scattering revealed an average particle size of 120 nm and a PDI (dispersity index) of 0.15 (Figure 2, D). The presence of neogambogic acid in the neogambogic acid fat bodies was further determined by TLC (thin-layer chromatography) and HPLC (high-performance liquid chromatography). The TLC assay involved adding an equal volume of methanol and two volumes of chloroform to the constructed neogambogic acid fat bodies to extract lipids. The organic phase was collected and dried with nitrogen to obtain total lipids. The total lipids were then added to 100 μl of chloroform, and 10 μl was loaded onto a silica gel plate. The plate was then developed in a solvent consisting of n-hexane: ether: glacial acetic acid (80:20:1 by volume) to separate triolein. The silica gel plate was then developed in a solvent consisting of chloroform:methanol:glacial acetic acid:water (volume ratio: 75:13:9:3) to separate EggPC, DOTAP, and NGA. TLC results indicated the presence of NGA signals in the fat bodies based on the position of the standard. In lane 4 (Blank-AD), no NGA signal was detected. In lane 5 (NGA-AD), an NGA signal was detected, but no DOTAP signal was detected. In lane 6 (NGA-C-AD), both NGA and DOTAP signals were detected. However, all of these fat bodies contained triolein signals (Figure 2, E). HPLC results showed that the elution time of NGA in the neogambogic acid fat bodies coincided with that of the free NGA standard in ethanol (Figure 2, F). At the same time, the Ztea potential results showed that NGA-C-AD was positively charged at about 60 mV, while Blank-AD and NGA-AD were not positively charged, at 0 mV and -20 mV, respectively (G in Figure 2). The above results indicate that the fat body containing neogamonic acid was successfully prepared with high purity and good uniformity.
实施例2新藤黄酸脂肪体具有抗肿瘤活性，效果显著优于游离的新藤黄酸Example 2 Neogambogic acid fat bodies have anti-tumor activity, and the effect is significantly better than free neogambogic acid
进一步我们检测了新藤黄脂肪体是否具有生物活性，能否杀伤肿瘤细胞。选择实施例1中制备的不同的新藤黄酸脂肪体处理直肠癌细胞CT26(图3中A)、乳腺癌细胞4T1(图3中B)和肝癌细胞Hepa1-6(图3中C)。结果显示相对于等体积的生理盐水组和Blank-AD组，随着NGA浓度的提高，NGA脂肪体杀伤癌细胞的效果越来越好更好，显著优于游离的NGA。此外，相较于带有NGA但不带阳离子的脂肪体(NGA-AD)，带有NGA又带有阳离子的脂肪体(NGA-C-AD)的杀伤各种癌细胞的效果最好。We further tested whether the new gamboge acid fat bodies have biological activity and whether they can kill tumor cells. The different new gamboge acid fat bodies prepared in Example 1 were selected to treat rectal cancer cells CT26 (Figure 3 A), breast cancer cells 4T1 (Figure 3 B) and liver cancer cells Hepa1-6 (Figure 3 C). The results showed that compared with the equal volume of normal saline group and Blank-AD group, with the addition of NGA As the concentration increased, the NGA fat bodies became increasingly effective at killing cancer cells, significantly outperforming free NGA. Furthermore, compared to fat bodies containing NGA but no cations (NGA-AD), fat bodies containing both NGA and cations (NGA-C-AD) were the most effective at killing various cancer cells.
细胞存活率的检测方法：将要处理的细胞以3000个细胞/孔铺在96孔板中，细胞过夜贴壁后，用图示的不同浓度药物处理上述细胞并孵育72小时。随后，加入含有10％CCK8的培养基替换原始培养基，孵育1小时后使用酶标仪读取450nm处的吸光度，通过以下公式计算细胞的存活率：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:
Cell Viability＝(Ae-Ab)/(Ac-Ab)*100％Cell Viability＝(Ae-Ab)/(Ac-Ab)*100%
其中，Ae表示含细胞的孔药物处理后的吸光度值。Wherein, Ae represents the absorbance value of the well containing cells after drug treatment.
Ac表示含细胞的孔未经药物处理的吸光度值。Ac represents the absorbance value of the wells containing cells without drug treatment.
Ab表示仅含培养基和CCK8试剂的空白孔吸光度值，用于校正背景噪音。Ab represents the absorbance value of blank wells containing only culture medium and CCK8 reagent, which is used to correct background noise.
实施例3新藤黄酸脂肪体抑制小鼠结直肠癌的生长，效果优于游离的新藤黄酸Example 3 Neogambogic acid fat bodies inhibit the growth of colorectal cancer in mice, and the effect is better than that of free neogambogic acid
前期体外细胞实验结果显示新藤黄酸脂肪体可以抑制结直肠癌细胞、乳腺癌细胞和肝癌细胞等多种癌细胞的生长，其抑制效果优于游离的新藤黄酸。我们进一步对比新藤黄酸脂肪体与游离的新藤黄酸在体内治疗小鼠结直肠癌的效果。Previous in vitro cell experiments have shown that neogambogic acid liposomes can inhibit the growth of various cancer cells, including colorectal cancer cells, breast cancer cells, and liver cancer cells, with an inhibitory effect superior to that of free neogambogic acid. We further compared the efficacy of neogambogic acid liposomes with that of free neogambogic acid in the treatment of colorectal cancer in mice in vivo.
首先将小鼠结直肠癌细胞CT26注射至BALB/c雌鼠背部进行皮下荷瘤，11天后随机分为5组，分别为生理盐水组、空载脂肪体组(Blank-AD)、游离的NGA、带有NGA但不带阳离子的脂肪体(NGA-AD)、带有NGA又带有阳离子的脂肪体(NGA-C-AD)，每组7只小鼠。每周给药1次，每次每只小鼠NGA的剂量都为10mg/kg。在给药期间分别检测小鼠肿瘤的大小及体重变化，结果如图4所示，相对于游离的NGA对肿瘤的抑制率为47.7％，而NGA-AD和NGA-C-AD抑制小鼠肿瘤的效果更好，肿瘤抑制率分别为59.8％和80.5％。新藤黄酸脂肪体处理后的小鼠的体重变化没有差异，血常规指标血红蛋白(HGB)、红细胞数量(RBC)和肾脏炎症指标肌酐(Cre)和也没有差异。但是，NGA-C-AD处理的小鼠的肝脏炎症指标天冬氨酸转氨酶(AST)显著减少，表明NGA-C-AD相较于NGA、NGA-AD具有更低的肝毒性。这些结果表明新藤黄酸脂肪体抑制小鼠结直肠癌的生长，效果显著优于游离的新藤黄酸，并且其安全性好，为其临床转化奠定了基础。First, mouse colorectal cancer cells CT26 were injected into the back of BALB/c female mice for subcutaneous tumor bearing. After 11 days, they were randomly divided into 5 groups, namely normal saline group, empty fat body group (Blank-AD), free NGA, fat body with NGA but without cations (NGA-AD), and fat body with NGA and cations (NGA-C-AD), with 7 mice in each group. The drug was administered once a week, and the dose of NGA for each mouse was 10 mg/kg each time. During the administration period, the size of the mouse tumor and the weight change were detected respectively. The results are shown in Figure 4. Compared with the free NGA, the inhibition rate of the tumor was 47.7%, while NGA-AD and NGA-C-AD had better effects on inhibiting mouse tumors, with tumor inhibition rates of 59.8% and 80.5%, respectively. There was no difference in the weight change of mice after treatment with neogamoic acid fat body. The blood routine indicators hemoglobin (HGB), red blood cell count (RBC) and renal There was no difference in creatinine (Cre) and creatinine (Cre), indicators of liver inflammation. However, aspartate aminotransferase (AST), an indicator of liver inflammation, was significantly reduced in mice treated with NGA-C-AD, indicating that NGA-C-AD has lower hepatotoxicity than NGA and NGA-AD. These results indicate that neogambogic acid fat bodies inhibit the growth of colorectal cancer in mice, with significantly better efficacy than free neogambogic acid, and its good safety profile lays the foundation for its clinical translation.
实施例4新藤黄酸脂肪体抑制小鼠肝癌的生长，效果优于游离的新藤黄酸Example 4 Neogambogic acid fat bodies inhibit the growth of mouse liver cancer, and the effect is better than free neogambogic acid
前期体外细胞实验结果显示新藤黄酸脂肪体可以抑制结直肠癌细胞、乳腺癌细胞和肝癌细胞等多种癌细胞的生长，其抑制效果优于游离的新藤黄酸。我们进一步对比新藤黄酸脂肪体与游离的新藤黄酸在体内治疗小鼠肝癌的效果。Previous in vitro cell experiments have shown that neogambogic acid liposomes can inhibit the growth of various cancer cells, including colorectal cancer cells, breast cancer cells, and liver cancer cells, with an inhibitory effect superior to that of free neogambogic acid. We further compared the efficacy of neogambogic acid liposomes with that of free neogambogic acid in treating liver cancer in mice in vivo.
我们构建了小鼠的肝癌肿瘤模型来探索新藤黄酸脂肪体的治疗效果。首先通过二乙基亚硝胺(DEN)联合四氯化碳构建C57BL/6小鼠肝癌模型，构建方法如下:配制DEN溶液：取少量DEN，用生理盐水配置成4mg/ml，按体重10μl/g腹腔注射。配制CCl4(20％)溶液：量取10ml CCl4，加入40ml玉米油配置成20％CCl4溶液，按体重5μl/g腹腔注射。2周龄小鼠腹腔注射一次DEN(40mg/kg)，小鼠饲养6周(8周龄小鼠)后，每周2次注射20％CCl4溶液，连续给药16周解剖观察肝脏，检查有无原发肝癌。若有，表明肝癌模型构建成功，然后将小鼠随机分为4组，分别为生理盐水组、空载脂肪体组(Blank-AD)、游离的NGA、带有NGA又带有阳离子的脂肪体(NGA-C-AD)，每组5只小鼠。每周给药2次，每次每只小鼠剂量为18mg/kg。相对于游离的NGA对肿瘤的抑制率为22.3％，NGA-C-AD抑制小鼠肿瘤的效果更好，肿瘤抑制率为76.2％。该结果与结直肠癌小鼠的结果一致，新藤黄酸脂肪体在小鼠体内具有良好的生物相容性。这些结果表明新藤黄酸脂肪体抑制小鼠肝癌的生长，效果显著优于游离的新藤黄酸，并且其安全性好，为其临床转化奠定了基础。We established a mouse liver cancer tumor model to explore the therapeutic effects of the new gambogic acid fat body. First, a C57BL/6 mouse liver cancer model was established by combining diethylnitrosamine (DEN) with carbon tetrachloride. The construction method is as follows: Prepare a DEN solution: Take a small amount of DEN, dilute it with normal saline to 4mg/ml, and inject it intraperitoneally at 10μl/g body weight. Prepare aCCl4 (20%) solution: Measure 10ml ofCCl4 , add 40ml of corn oil to make a 20% CCl4 solution, and inject it intraperitoneally at 5μl/g body weight. 2-week-old mice were intraperitoneally injected with DEN (40mg/kg) once. After the mice were raised for 6 weeks (8-week-old mice), they were injected with 20%CCl4 solution twice a week. After 16 weeks of continuous administration, the livers were dissected and observed to check for primary liver cancer. If yes, it indicates that the liver cancer model was successfully constructed, and then the mice were randomly divided into 4 groups, namely normal saline group, empty fat body group (Blank-AD), free NGA, and fat body with NGA and cations (NGA-C-AD), with 5 mice in each group. The drug was administered twice a week, with a dose of 18 mg/kg per mouse each time. Compared with the tumor inhibition rate of 22.3% of free NGA, NGA-C-AD had a better effect in inhibiting mouse tumors, with a tumor inhibition rate of 76.2%. This result is consistent with the results of colorectal cancer mice, and the new gambogic acid fat body has good biocompatibility in mice. These results show that the new gambogic acid fat body inhibits the growth of mouse liver cancer, and the effect is significantly better than that of free new gambogic acid, and it has good safety, which lays the foundation for its clinical transformation.
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention. It should be noted that for ordinary technicians in this technical field, several improvements and refinements can be made without departing from the principles of the present invention. These improvements and modifications should also be considered as the protection scope of the present invention.
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| CN112546201A (en)* | 2019-09-26 | 2021-03-26 | 中国科学院生物物理研究所 | Application of artificial lipoprotein particle apoA-IV fat body in treatment and/or prevention of diabetes |
| CN111057136A (en)* | 2019-12-30 | 2020-04-24 | 中国科学院生物物理研究所 | A fat body and its application in evaluating the interaction between a protein to be tested and lipid droplets |
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