技术领域technical field
本发明属于医药领域，涉及一种肝靶向长循环脂质体及其制备方法和用途，具体涉及一种两亲性甘草次酸衍生物的合成方法，以及含有这种衍生物的脂质体(liposome)及其作为药物、生物活性物质、示踪物、诊断试剂或影像增强剂的肝靶向长循环载体的应用。The invention belongs to the field of medicine, and relates to a liver-targeted long-circulation liposome and its preparation method and application, in particular to a synthesis method of an amphipathic glycyrrhetinic acid derivative, and a liposome containing the derivative (liposome) and its use as a liver-targeted long-circulating carrier for drugs, bioactive substances, tracers, diagnostic reagents or image enhancers.
背景技术Background technique
我国是世界上肝脏疾病的高发区，癌症发病率处于世界前列，特别是肝癌、食道癌和胃癌的发病率为世界第一。当前，据血清流行病学调查，我国人群的乙肝表面抗原(HBsAg)携带率为10％，约1.3亿人，其中1/4的人最终将发展为慢性肝病，包括慢性肝炎、肝硬化和肝癌，丙肝病毒感染者也已超千万人。然而，面对如此庞大的患者人群，治疗现状却不容乐观。肝脏的病变不仅危害肝脏，同时也会因代谢和解毒功能受损而影响心脏、大脑、消化系统、内分泌和免疫力，引发各种心脑血管疾病和肿瘤，因此预防和治疗肝脏病变对于人体极其重要。对于早期肝癌患者来说癌细胞并未扩散到淋巴结和身体的其它部位，手术切除治疗是最为有效的治疗手段。然而肝癌的发病较隐匿，早期患者无明显症状，一旦出现症状前往医院就诊时，其病程大多已进入中、晚期，错过了手术治疗的最佳时期。此时即使手术切除，治疗成功率也仅为20％～30％。因此，实现肝癌的早期高灵敏检测是手术治疗能否成功的关键因素。这对于显像学的发展提出了更高的要求。而对于肝炎、肝硬化以及中晚期肝癌患者：非手术治疗，尤其是药物治疗就成为了综合治疗的重要手段之一。化疗是目前除了手术治疗之外最主要的治疗方法。但传统的化疗方式是通过常规途径给药，当达到一定的血药浓度后药物在全身分布，缺乏选择性，在杀伤癌细胞的同时还会对其它正常脏器产生严重的毒副作用，不仅影响疗效也给病人带来巨大痛苦。由此可见传统治疗方法滞后，治疗效果不好，不少治疗方法甚至是边治肝边伤肝，使肝病治疗陷入了一个久治不愈、久药不果的怪圈。my country is an area with a high incidence of liver diseases in the world, and the incidence of cancer is in the forefront of the world, especially the incidence of liver cancer, esophageal cancer and gastric cancer is the highest in the world. At present, according to the serological epidemiological survey, the carrier rate of hepatitis B surface antigen (HBsAg) in the Chinese population is 10%, about 130 million people, of which 1/4 will eventually develop chronic liver diseases, including chronic hepatitis, cirrhosis and liver cancer , Hepatitis C virus infection has also exceeded ten million people. However, in the face of such a large patient population, the treatment status is not optimistic. Liver lesions not only endanger the liver, but also affect the heart, brain, digestive system, endocrine and immunity due to impaired metabolism and detoxification, causing various cardiovascular and cerebrovascular diseases and tumors. Therefore, prevention and treatment of liver lesions are extremely important to the human body. important. Surgical resection is the most effective treatment for patients with early-stage liver cancer that has not spread to the lymph nodes and other parts of the body. However, the onset of liver cancer is relatively hidden, and patients in the early stage have no obvious symptoms. Once symptoms appear and go to the hospital for treatment, most of the disease course has entered the middle or late stage, and the best period for surgical treatment has been missed. At this time, even surgical resection, the success rate of treatment is only 20% to 30%. Therefore, the realization of early and highly sensitive detection of liver cancer is a key factor for the success of surgical treatment. This puts forward higher requirements for the development of imaging science. For patients with hepatitis, liver cirrhosis, and advanced liver cancer: non-surgical treatment, especially drug treatment, has become one of the important means of comprehensive treatment. Chemotherapy is currently the main treatment method besides surgery. However, traditional chemotherapy is administered through conventional routes. When a certain blood drug concentration is reached, the drug is distributed throughout the body, which lacks selectivity. While killing cancer cells, it will also cause serious toxic and side effects to other normal organs, which not only affects The curative effect also brings great pain to the patient. It can be seen that the traditional treatment methods are lagging behind, and the treatment effect is not good. Many treatment methods even treat the liver while injuring the liver.
因此改变化疗的给药途径、变换药物剂型等各种各样的尝试势在必行，也从未间断。靶向给药系统可以将药物运送到指定的靶器官或靶组织释放，与传统的化疗相比，可以提高药物在靶部位的药理作用强度，明显降低药物毒副作用，提高治疗效果。Therefore, various attempts such as changing the administration route of chemotherapy and changing drug dosage forms are imperative and have never stopped. The targeted drug delivery system can deliver the drug to the designated target organ or target tissue for release. Compared with traditional chemotherapy, it can increase the pharmacological effect of the drug on the target site, significantly reduce the side effects of the drug, and improve the therapeutic effect.
随着对细胞表面结构功能认识的不断深入，近年来受体介导的肝靶向递送系统成为研究热点。它是采用物理或化学的方法将特定的配基引入药物载体，通过配基与肝脏细胞膜上的受体发生特异性相互作用，将药物、基因等化学物质选择性地输送至肝脏，提高负载物在肝脏部位的浓度，延长其半衰期，从而达到减少用药剂量和给药次数，降低药物毒副作用，减少对其它脏器的损伤，提高药效。With the deepening understanding of cell surface structure and function, receptor-mediated liver-targeted delivery system has become a research hotspot in recent years. It uses physical or chemical methods to introduce specific ligands into drug carriers, and through specific interactions between ligands and receptors on the liver cell membrane, chemical substances such as drugs and genes are selectively delivered to the liver, increasing the load capacity. The concentration in the liver prolongs its half-life, thereby reducing the dosage and frequency of administration, reducing the toxic and side effects of the drug, reducing the damage to other organs, and improving the efficacy of the drug.
纳米颗粒和细胞的相互作用包括细胞吞噬、被动靶向、主动靶向等。被动靶向指的是载药微粒进入体内后，由于肿瘤与正常组织间血管密度及渗透性的差异或被巨噬细胞作为外界异物吞噬的自然倾向而产生的体内分布特征。一般认为：被动靶向的微粒经静脉注射后其在体内的分布首先取决于粒径的大小：小于100nm的纳米囊或纳米球可缓慢积集骨髓；100～200nm的纳米粒子可富集于实体肿瘤部位；0.2～0.3μm的粒子一般被肝脾中的巨噬细胞摄取；大于7μm的微粒通常被肺毛细血管床截留进入肺组织或肺气泡。The interaction between nanoparticles and cells includes cell phagocytosis, passive targeting, active targeting, etc. Passive targeting refers to the in vivo distribution characteristics of drug-loaded particles after entering the body due to the difference in blood vessel density and permeability between tumors and normal tissues, or the natural tendency of macrophages to be swallowed by macrophages as foreign bodies. It is generally believed that the distribution of passively targeted microparticles in the body after intravenous injection firstly depends on the particle size: nanocapsules or nanospheres smaller than 100 nm can slowly accumulate in the bone marrow; nanoparticles of 100-200 nm can be enriched in solid Tumor site; particles of 0.2-0.3 μm are generally taken up by macrophages in the liver and spleen; particles larger than 7 μm are usually trapped by the pulmonary capillary bed and enter the lung tissue or alveoli.
肝脏拥有人体内数量最多的吞噬细胞——枯否细胞(KCs)，约占吞噬细胞总数的80％。研究表明，具有以下特征的颗粒会被迅速清除，并在肝脾等器官累积，从而产生一定被动靶向功效：①拥有较大的粒径(直径>200nm)；②非球形颗粒；③表面具有较强疏水性；④表面具有高密度电荷。由此可见，被动肝靶向给药体系主要依赖于体内的单核吞噬细胞系统(MPS)。因此利用MPS的吞噬功能就成为了肝靶向给药的有效途径。尽管MPS靶向对于治疗肝肿瘤具有一定的促进作用，但众所周知，恶性肝肿瘤往往发生在肝实质细胞，而MPS会将大量药物聚集在枯否细胞中，这就造成大量药物无法直接作用于肿瘤部位，影响治疗效果。因此开发肝实质细胞靶向给药系统就显得尤为重要(王蔚，袁直，“肝靶向纳米给药系统的最新研究进展”，高分子通报，2013，1：137～154)。The liver has the largest number of phagocytes in the human body - Kupffer cells (KCs), accounting for about 80% of the total number of phagocytes. Studies have shown that particles with the following characteristics will be cleared quickly and accumulate in organs such as the liver and spleen, thereby producing a certain passive targeting effect: ①Has a large particle size (diameter>200nm); ②Non-spherical particles; ③The surface has Strong hydrophobicity; ④The surface has high density charges. It can be seen that the passive liver-targeted drug delivery system mainly depends on the mononuclear phagocyte system (MPS) in vivo. Therefore, using the phagocytic function of MPS has become an effective way for liver-targeted drug delivery. Although MPS targeting can promote the treatment of liver tumors, it is well known that malignant liver tumors often occur in hepatic parenchymal cells, and MPS will accumulate a large number of drugs in Kupffer cells, which prevents a large number of drugs from directly acting on tumors site, affecting the therapeutic effect. Therefore, it is particularly important to develop a liver parenchymal cell-targeted drug delivery system (Wang Wei, Yuan Zhi, "The latest research progress in liver-targeted nano drug delivery system", Polymer Bulletin, 2013, 1: 137-154).
主动靶向给药系统主要包括三个部分：靶向基团、载体以及药物。主动靶向给药就是载体将药物通过局部给药或全身血液循环选择性地富集于靶器官、靶组织、靶细胞或细胞内结构中。为了降低MPS系统对主动靶向效率的影响，首先应保证药物载体能逃离MPS的吞噬，并实现在血液内的长效循环。因此，一般均需对纳米粒子表面进行亲水改性并控制其粒径在100～200nm之间(亦有文献报道为50～150nm)。由于PEG具有高亲水性、高体积排阻效应以及低毒性已经成为目前最常用的亲水改性试剂。The active targeted drug delivery system mainly includes three parts: targeting group, carrier and drug. Active targeted drug delivery means that the carrier selectively enriches the drug in the target organ, target tissue, target cell or intracellular structure through local administration or systemic blood circulation. In order to reduce the impact of the MPS system on the active targeting efficiency, it is first necessary to ensure that the drug carrier can escape the phagocytosis of the MPS and achieve long-term circulation in the blood. Therefore, it is generally necessary to carry out hydrophilic modification on the surface of nanoparticles and control the particle size between 100-200 nm (also reported as 50-150 nm in literature). Due to its high hydrophilicity, high size exclusion effect and low toxicity, PEG has become the most commonly used hydrophilic modification reagent.
脂质体作为一种抗肿瘤化疗药物的载体，也是最理想的非病毒基因载体，是一类由两亲性分子(多为磷脂)形成的具有类似细胞膜双分子层结构的囊泡，可以将多种物质(药物、核酸等)转运至动物、植物和微生物等多种类型的细胞中，使被包载物的生物学活性不被破坏，而且还可以提高传递效率，因而在生物、化学、医药等领域得到广泛研究和应用。脂质体本身具有被动靶向的性质，经静脉注射给药之后能选择性地分布于肝脏、脾脏、骨髓等内皮网状系统(RES)丰富的组织器官中。但脂质体在肝脏中主要是被肝脏的非实质细胞组织所内化吞噬入细胞内而吸收，只有少量能够被肝脏实质细胞所吸收。肝实质细胞是肝脏中数量最多的细胞，约占肝脏总细胞数的60％～70％，有关肝的大多数病变如肝癌、肝炎、肝硬化等多发生于此，是肝靶向给药系统理想的靶标之一，因此只有有效提高药物对病灶组织的靶向性，才能提高载药脂质体在肝脏实质细胞中的吸收量，从而真正发挥药效。As a carrier of anti-tumor chemotherapy drugs, liposome is also the most ideal non-viral gene carrier. It is a vesicle with a similar cell membrane bilayer structure formed by amphiphilic molecules (mostly phospholipids). Various substances (drugs, nucleic acid, etc.) Medicine and other fields have been widely researched and applied. Liposome itself has the nature of passive targeting, and can be selectively distributed in tissues and organs rich in endothelial reticular system (RES) such as liver, spleen, and bone marrow after intravenous injection. However, liposomes in the liver are mainly absorbed by the non-parenchymal cells of the liver, and only a small amount can be absorbed by the parenchymal cells of the liver. Hepatic parenchymal cells are the most numerous cells in the liver, accounting for about 60% to 70% of the total number of liver cells. Most liver-related diseases such as liver cancer, hepatitis, and cirrhosis often occur here. They are liver-targeted drug delivery systems. One of the ideal targets. Therefore, only by effectively improving the targeting of the drug to the lesion tissue can the absorption of the drug-loaded liposome in the liver parenchymal cells be improved, so as to truly exert the drug effect.
肝实质细胞表面含有多种受体：如去唾液酸糖蛋白受体可以识别半乳糖乳糖等配体，甘草酸、甘草次酸受体可以识别甘草酸以及甘草次酸等。利用这些受体配体间特异性的相互作用，研究人员研发出一系列高效的肝靶向给药系统。目前已见报导的配基有去唾液酸糖蛋白、乳糖酸、半乳糖化配体、无唾液酸胎球蛋白以及豆甾醇糖苷等。其中去唾液酸糖蛋白受体能专一识别末端带有半乳糖残基或乙酰半乳糖胺残基的寡糖或寡糖蛋白。胆酸是胆汁的主要成分，在体内具有特殊的转运系统，可被肝脏特异性吸收。胆酸盐介导的肝靶向给药系统是通过肝细胞膜上的Na+依赖性转运系统(NTCP)及Na+非依赖性转运系统(OATP)来实现的，以胆酸为药物载体，不但能够实现药物的肝靶向性，减少毒副作用，还能够提高药物的口服生物利用度。甘露糖受体是分子量为175000的跨膜蛋白，能与含有甘露糖配基的物质特异性识别结合，广泛存在于肝细胞表面。此外研究比较多的还有透明质酸受体、胰岛素受体等。与这些配体物质相比，能与甘草酸、甘草次酸受体特异性作用的甘草酸和甘草次酸配体有着独特的优势。The surface of liver parenchymal cells contains a variety of receptors: for example, asialoglycoprotein receptors can recognize ligands such as galactose lactose, and glycyrrhizic acid and glycyrrhetinic acid receptors can recognize glycyrrhizic acid and glycyrrhetinic acid. Using the specific interactions between these receptor ligands, researchers have developed a series of highly efficient liver-targeted drug delivery systems. Ligands that have been reported so far include asialoglycoprotein, lactobionic acid, galactosylated ligands, asialofetuin, and stigmasterol glycosides. Among them, the asialoglycoprotein receptor can specifically recognize oligosaccharides or oligosaccharide proteins with galactose residues or acetylgalactosamine residues at the end. Bile acid is the main component of bile. It has a special transport system in the body and can be specifically absorbed by the liver. The bile salt-mediated liver-targeted drug delivery system is realized through the Na+ -dependent transport system (NTCP) and the Na+ -independent transport system (OATP) on the liver cell membrane. Using bile acid as a drug carrier, not only The liver targeting of the drug can be realized, the toxic and side effects can be reduced, and the oral bioavailability of the drug can also be improved. Mannose receptors are transmembrane proteins with a molecular weight of 175,000, which can specifically recognize and bind substances containing mannoglycosides, and are widely present on the surface of liver cells. In addition, there are more studies on hyaluronic acid receptors and insulin receptors. Compared with these ligand substances, glycyrrhizic acid and glycyrrhetinic acid ligands that can specifically interact with glycyrrhizic acid and glycyrrhetinic acid receptors have unique advantages.
甘草是中国自古以来就应用广泛的一种重要传统中药，根据现代的甘草药理学、体内代谢学研究可知，当甘草酸口服之后，经胃酸水解或者由肝脏中β-葡萄糖醛酸酶酶解可以得到甘草酸类药物中主要发挥药力活性作用的五环三萜类化合物——甘草次酸，所以甘草次酸是甘草中最终起作用的成分。甘草次酸来源于豆科植物甘草的根及根茎，廉价易得，安全性高，具有多种药理活性，如抗炎、抗菌、抗肿瘤、抗溃疡、抗病毒性肝炎及保肝护肝等。除了较强的治疗炎症的效果外，还具有激活免疫系统活性、抑制肿瘤细胞增殖和转移、抗心律失常、抗病毒、提高内耳听力抗缺氧等多种治疗作用。研究表明，肝细胞表面存在大量甘草次酸受体，甘草次酸对肝细胞有特异识别和吸附能力，与该位点的结合呈可饱和性、高度特异性，能有效的发挥其在肝脏疾病中的治疗作用，目前在临床上较多的用于慢性肝炎以及肝癌的治疗，例如甘草次酸修饰的纳米粒子用于肝靶向药物传递【TIANQ，ZHANGCN，WANGXH，etal.“Glycyrrhetinicacid-modifiedchitosan/poly(ethyleneglycol)nanoparticlesforliver-targeteddeliver”[J]Biomaterials，2010，31(17)：4748-4756】。Licorice is an important traditional Chinese medicine that has been widely used in China since ancient times. According to modern licorice pharmacology and in vivo metabolism studies, after oral administration, licorice can be hydrolyzed by gastric acid or enzymatically hydrolyzed by β-glucuronidase in the liver. Glycyrrhetinic acid, a pentacyclic triterpenoid compound that mainly exerts pharmacological activity in glycyrrhizic acid drugs, is obtained, so glycyrrhetinic acid is the final active ingredient in licorice. Glycyrrhetinic acid is derived from the roots and rhizomes of licorice, a leguminous plant. It is cheap, easy to obtain, and has high safety. It has various pharmacological activities, such as anti-inflammatory, antibacterial, anti-tumor, anti-ulcer, anti-viral hepatitis, and liver protection. . In addition to the strong effect of treating inflammation, it also has various therapeutic effects such as activating the activity of the immune system, inhibiting the proliferation and metastasis of tumor cells, anti-arrhythmia, anti-virus, improving inner ear hearing and anti-hypoxia. Studies have shown that there are a large number of glycyrrhetinic acid receptors on the surface of liver cells. Glycyrrhetinic acid has specific recognition and adsorption capabilities for liver cells, and the binding to this site is saturable and highly specific, which can effectively play its role in liver diseases. Therapeutic role in the treatment of chronic hepatitis and liver cancer in clinical practice, such as glycyrrhetinic acid modified nanoparticles for liver-targeted drug delivery [TIANQ, ZHANGCN, WANGXH, et al. "Glycyrrhetinic acid-modifiedchitosan/ poly(ethyleneglycol) nanoparticles for liver-targeted deliver” [J] Biomaterials, 2010, 31(17): 4748-4756].
近年来，已有有关表面经甘草次酸修饰的脂质体用于药物肝靶向的传输以及治疗的报道【MaoSJ，BiYQ，JinH，etal.“Preparation，characterizationanduptakebyprimaryculturedrathepatocytesofliposomessurface-modifiedwithglycyrrhetinicacid”.DiePharmazie，2007，62：614-619】。ChenZP等人【ChenZP，XiaoL，LiuD，etal.“Synthesisofanovelpolymercholesterol-poly(ethyleneglycol)2000-glycyrrhetinicacid(Chol-PEG-GA)anditsapplicationinbrucineliposome”[J].JournalofAppliedPolymerScience，2012，124(6)：4554-4563】将胆固醇、聚乙二醇和甘草次酸偶联以得到两亲性化合物Chol-PEG-GA，并将其用于制备甘草次酸修饰的长循环脂质体(Chol-PEG-GALiposome，CPGL)来包载二甲马钱子碱，再将该载药脂质体静脉注射入小鼠体内进行组织分布研究，以传统脂质体为对照。实验结果显示，载药CPGL脂质体的药时曲线下面积(AreaUnderCurve，AUC)以及平均驻留时间(MeanRetentionTime，MRT)分别为传统脂质体的2.31倍和2.11倍；另外，使用甘草次酸修饰的载二甲马钱子碱脂质体CPGL注射后，药物在小鼠肝脏组织中的浓度要比使用传统脂质体的小鼠肝脏组织浓度高七倍左右，这表明甘草次酸修饰的长循环脂质体具有显著的肝靶向性和延长药物释放时间的效果。In recent years, there have been reports about liposomes modified with glycyrrhetinic acid on the surface for liver-targeted drug delivery and treatment [MaoSJ, BiYQ, JinH, et al. "Preparation, characterization and uptake by primary culture drafts of liposomes surface-modified with glycyrrhetinic acid". :614-619]. ChenZP et al [ChenZP, XiaoL, LiuD, et al. "Synthesis of novel polymercholesterol-poly(ethyleneglycol) 2000-glycyrrhetinicacid(Chol-PEG-GA) and its application in brucineliposome" [J]. Journal of Applied PolymerScience, 2012, 124(6): 4553] 4-45 , polyethylene glycol and glycyrrhetinic acid were coupled to obtain the amphiphilic compound Chol-PEG-GA, and it was used to prepare glycyrrhetinic acid-modified long-circulating liposomes (Chol-PEG-GALiposome, CPGL) to entrap Dimethyl strychnine, and then inject the drug-loaded liposome intravenously into mice for tissue distribution research, with traditional liposome as a control. The experimental results show that the area under the drug-time curve (AreaUnderCurve, AUC) and mean residence time (MeanRetentionTime, MRT) of the drug-loaded CPGL liposomes are 2.31 times and 2.11 times that of the traditional liposomes respectively; in addition, using glycyrrhetinic acid After the modified dimethyl-strychnine-loaded liposome CPGL was injected, the concentration of the drug in the liver tissue of the mouse was about seven times higher than that of the mouse liver tissue using the traditional liposome, which indicated that the glycyrrhetinic acid-modified Long-circulating liposomes have significant liver-targeting and prolonged drug release effects.
中国专利CN200810156141.7公开了甘草次酸前体脂质体及其制备方法，该专利是将甘草次酸直接作为药物包载在脂质体脂双层中，并引入了聚乙二醇以起到长循环作用。Chinese patent CN200810156141.7 discloses glycyrrhetinic acid proliposome and its preparation method. This patent is to directly entrap glycyrrhetinic acid in the liposome lipid bilayer as a drug, and introduce polyethylene glycol to to long-term circulation.
中国专利CN201010228799.1公开了甘草次酸修饰磷脂和胆固醇作为脂质体膜材使用，合成肝靶向脂质体、胶束和复合物的制法，期望达到肝靶向长循环的作用；但是该专利可能存在的问题：一是在合成其中一个物质甘草次酸修饰聚乙二醇化胆固醇时，先将甘草次酸与聚乙二醇反应而未对甘草次酸上的羟基进行保护，这样反应结束后两端都有羟基，之后再与胆固醇反应，这样就存在以下两种可能：(1)反应可能生成胆固醇-甘草次酸-聚乙二醇，甘草次酸的两端均被键连，这样作为膜材组成脂质体时，甘草次酸很可能被埋在了脂双层中，很难真正起到肝靶向作用；(2)该反应也可能生成甘草次酸-聚乙二醇-胆固醇，这个物质才能真正起到肝靶向作用。但是该专利中并没有具体说明制备肝靶向脂质体所用的是哪一种化合物。二是该专利实施例5在制备脂质体时是先制备空白肝靶向脂质体，然后再与绿荧光蛋白质粒溶液孵育，虽然脂质体具有一定的流动性，也很可能导致大量质粒游离在脂质体之外，很多肝靶向脂质体还是空白脂质体，治疗效果会大打折扣。实施例6中是先制备空白载基因脂质体，再加入甘草次酸修饰聚乙二醇化的胆固醇进行孵育，以起到肝靶向作用，这同样存在甘草次酸修饰聚乙二醇化的胆固醇无法顺利嵌入脂双层中的问题，很难真正合成肝靶向脂质体。Chinese patent CN201010228799.1 discloses the use of glycyrrhetinic acid modified phospholipids and cholesterol as liposome membrane materials to synthesize liver-targeted liposomes, micelles and complexes, expecting to achieve long-term liver-targeted circulation; but Possible problems in this patent: First, when synthesizing one of the substances, glycyrrhetinic acid to modify PEGylated cholesterol, glycyrrhetinic acid is first reacted with polyethylene glycol without protecting the hydroxyl groups on glycyrrhetinic acid. After the end, there are hydroxyl groups at both ends, and then react with cholesterol, so there are two possibilities: (1) The reaction may generate cholesterol-glycyrrhetinic acid-polyethylene glycol, and both ends of glycyrrhetinic acid are bonded, In this way, when forming liposomes as a membrane material, glycyrrhetinic acid is likely to be buried in the lipid bilayer, and it is difficult to really play a liver targeting role; (2) This reaction may also generate glycyrrhetinic acid-polyethylene glycol -Cholesterol, this substance can really play a role in liver targeting. However, the patent does not specify which compound is used to prepare the liver-targeted liposome. Second, when liposomes are prepared in Example 5 of this patent, blank liver-targeted liposomes are prepared first, and then incubated with the green fluorescent protein particle solution. Free from the liposomes, many liver-targeted liposomes are still blank liposomes, and the therapeutic effect will be greatly reduced. In Example 6, blank gene-carrying liposomes were prepared first, and then glycyrrhetinic acid-modified PEGylated cholesterol was added for incubation to play a liver-targeting role. Glycyrrhetinic acid-modified PEGylated cholesterol also existed The problem of not being able to embed smoothly in the lipid bilayer makes it difficult to truly synthesize liver-targeting liposomes.
发明内容Contents of the invention
为了克服现有技术存在的不足，本发明采用甘草次酸作为肝实质细胞的靶向配体，共价连接具有长循环作用的聚乙二醇，并在聚乙二醇另一端共价连接可以定位在脂双层中的硬脂酸，制备一种两亲性甘草次酸衍生物——乙酰甘草次酸-聚乙二醇-硬脂酸(AGA-PEG2000-ST)，含有该化合物的脂质体同时具备了肝实质细胞靶向和长循环作用，采用常规的脂质体组分和制备方法可得到包载药物或造影剂、临床所用试剂的脂质体，可望应用于临床。In order to overcome the deficiencies in the prior art, the present invention adopts glycyrrhetinic acid as the targeting ligand of hepatic parenchymal cells, covalently connects polyethylene glycol with long-term circulation effect, and covalently connects the other end of polyethylene glycol to Stearic acid located in the lipid bilayer, prepare an amphiphilic glycyrrhetinic acid derivative - acetylglycyrrhetinic acid-polyethylene glycol-stearic acid (AGA-PEG2000 -ST), containing the compound Liposomes have both liver parenchymal cell targeting and long-term circulation effects. Using conventional liposome components and preparation methods, liposomes can be obtained that contain drugs, contrast agents, and clinically used reagents, and are expected to be used in clinical practice.
本发明的目的是合成一种经济、稳定、实用性强的肝靶向长循环脂质体分子构件，以用作一系列亲疏水药物、基因、造影剂、示踪物、诊断试剂等的载体，由于采用本发明提供的分子构件所制备的脂质体具有长循环肝靶向的性质，从而可以提高包载物对肝脏实质细胞的靶向性，降低毒副作用，提高疗效。The purpose of the present invention is to synthesize an economical, stable, and practical liver-targeted long-circulation liposome molecular component to be used as a carrier for a series of hydrophilic and hydrophobic drugs, genes, contrast agents, tracers, diagnostic reagents, etc. Because the liposome prepared by using the molecular component provided by the present invention has the property of long-circulation liver targeting, it can improve the targeting of the encapsulated substance to the liver parenchymal cells, reduce toxic and side effects, and improve curative effect.
本发明利用酰化反应对甘草次酸C3位的羟基进行保护，聚乙二醇一端的羟基和甘草次酸C30位羧基反应生成酯，而聚乙二醇的另一端的羟基则与硬脂酸反应生成酯。具体地，首先，由甘草次酸(GA)与乙酰氯(摩尔比为1：5～10)反应，以与甘草次酸1～2倍摩尔量的碳酸钾或三乙胺作为缚酸剂，得到乙酰甘草次酸(AGA)；接着乙酰甘草次酸在脱水剂二环己基碳二亚胺(DCC)和催化剂二甲氨基吡啶(DMAP)的存在下，控制反应条件，与聚乙二醇单端反应，得到乙酰甘草次酸-聚乙二醇；然后将硬脂酸用过量的二氯亚砜进行酰氯化，产物纯化后，与乙酰甘草次酸-聚乙二醇进行反应，得到乙酰甘草次酸-聚乙二醇-硬脂酸。The present invention uses acylation reaction to protect the hydroxyl group at the C3 position of glycyrrhetinic acid, the hydroxyl group at one end of polyethylene glycol reacts with the carboxyl group at the C30 position of glycyrrhetinic acid to form an ester, and the hydroxyl group at the other end of polyethylene glycol reacts with stearic acid The reaction produces an ester. Specifically, firstly, react glycyrrhetinic acid (GA) with acetyl chloride (molar ratio 1:5-10), and use potassium carbonate or triethylamine in an amount 1-2 times the molar amount of glycyrrhetinic acid as an acid-binding agent, Obtain acetylglycyrrhetinic acid (AGA); then acetylglycyrrhetinic acid in the presence of dehydrating agent dicyclohexylcarbodiimide (DCC) and catalyst dimethylaminopyridine (DMAP), control the reaction conditions, and polyethylene glycol mono terminal reaction to obtain acetylglycyrrhetinic acid-polyethylene glycol; then stearic acid is acid-chlorinated with excess thionyl chloride, and after the product is purified, it is reacted with acetylglycyrrhetinic acid-polyethylene glycol to obtain acetylglycyrrhizin Hypoglycerol-Polyethylene Glycol-Stearic Acid.
本发明所提供的技术方案具体如下：The technical scheme provided by the present invention is specifically as follows:
一种甘草次酸衍生物，具有式I所示的结构：A glycyrrhetinic acid derivative has a structure shown in formula I:
乙酰甘草次酸-聚乙二醇-硬脂酸Acetylglycyrrhetinic acid-polyethylene glycol-stearic acid
一种制备上述甘草次酸衍生物的方法，包括以下步骤：A method for preparing the above-mentioned glycyrrhetinic acid derivatives, comprising the following steps:
(1)乙酰甘草次酸的合成：将甘草次酸溶解在二氯甲烷中，然后加入缚酸剂，在冰浴条件下缓慢滴加乙酰氯，然后室温搅拌反应8～24小时，反应结束后，纯化，即得到乙酰甘草次酸；所述缚酸剂为碳酸钾或三乙胺，所述缚酸剂的摩尔量为甘草次酸的1～2倍，所述乙酰氯的摩尔量为甘草次酸的5～10倍；(1) Synthesis of acetylglycyrrhetinic acid: Dissolve glycyrrhetinic acid in methylene chloride, then add an acid-binding agent, slowly add acetyl chloride dropwise under ice-bath conditions, then stir and react at room temperature for 8 to 24 hours, after the reaction , purify to obtain acetylglycyrrhetinic acid; the acid-binding agent is potassium carbonate or triethylamine, the molar weight of the acid-binding agent is 1 to 2 times that of glycyrrhetinic acid, and the molar weight of the acetyl chloride is licorice 5 to 10 times that of hypoacid;
(2)乙酰甘草次酸-聚乙二醇的合成：将步骤(1)中得到的乙酰甘草次酸溶解在二氯甲烷中，制备乙酰甘草次酸的二氯甲烷溶液；同时将聚乙二醇、二环己基碳二亚胺、二甲氨基吡啶溶解在二氯甲烷中，边搅拌边缓慢滴加到乙酰甘草次酸的二氯甲烷溶液中，搅拌回流18～30小时，反应结束后，纯化，即得到乙酰甘草次酸-聚乙二醇；其中，所述聚乙二醇的摩尔量为乙酰甘草次酸的1～1.25倍，所述二环己基碳二亚胺的摩尔量为乙酰甘草次酸的1～1.2倍，所述二甲氨基吡啶的摩尔量为乙酰甘草次酸的0.02～0.1倍；(2) Synthesis of acetylglycyrrhetinic acid-polyethylene glycol: the acetylglycyrrhetinic acid obtained in step (1) is dissolved in dichloromethane to prepare the dichloromethane solution of acetylglycyrrhetinic acid; Alcohol, dicyclohexylcarbodiimide, and dimethylaminopyridine were dissolved in dichloromethane, and slowly added dropwise to the dichloromethane solution of acetylglycyrrhetinic acid while stirring, and stirred and refluxed for 18 to 30 hours. After the reaction, Purify to obtain acetylglycyrrhetinic acid-polyethylene glycol; wherein, the molar weight of polyethylene glycol is 1 to 1.25 times that of acetyl glycyrrhetinic acid, and the molar weight of dicyclohexylcarbodiimide is acetyl 1 to 1.2 times that of glycyrrhetinic acid, and the molar weight of dimethylaminopyridine is 0.02 to 0.1 times that of acetylglycyrrhetinic acid;
(3)乙酰甘草次酸-聚乙二醇-硬脂酸的合成：向硬脂酰氯中加入二氯甲烷、二甲氨基吡啶、三乙胺；同时将乙酰甘草次酸-聚乙二醇溶解在二氯甲烷中，冰浴条件下缓慢滴加入烧瓶，搅拌回流18～30小时，纯化，干燥，即得到上述甘草次酸衍生物；所述硬脂酰氯的摩尔量为乙酰甘草次酸-聚乙二醇的1～2倍，所述三乙胺的摩尔量为乙酰甘草次酸-聚乙二醇的1～2倍，所述二甲氨基吡啶的摩尔量为乙酰甘草次酸-聚乙二醇摩尔量的0.02～0.1倍。(3) Synthesis of acetylglycyrrhetinic acid-polyethylene glycol-stearic acid: add methylene chloride, dimethylaminopyridine, triethylamine to stearyl chloride; simultaneously dissolve acetylglycyrrhetinic acid-polyethylene glycol In dichloromethane, slowly drop into the flask under ice-bath conditions, stir and reflux for 18-30 hours, purify and dry to obtain the above-mentioned glycyrrhetinic acid derivatives; the molar weight of the stearyl chloride is acetylglycyrrhetinic acid-poly 1 to 2 times that of ethylene glycol, the molar weight of said triethylamine is 1 to 2 times that of acetylglycyrrhetinic acid-polyethylene glycol, and the molar weight of said dimethylaminopyridine is 1 to 2 times that of acetyl glycyrrhetinic acid-polyethylene glycol. 0.02 to 0.1 times the molar weight of diol.
步骤(2)所述聚乙二醇的摩尔量为乙酰甘草次酸的1.25倍。The molar weight of polyethylene glycol in step (2) is 1.25 times that of acetylglycyrrhetinic acid.
步骤(2)所述的搅拌回流时间为24小时。The stirring reflux time described in step (2) is 24 hours.
步骤(3)所述硬脂酰氯的摩尔量为乙酰甘草次酸-聚乙二醇的2倍。The molar weight of stearyl chloride described in step (3) is twice that of acetylglycyrrhetinic acid-polyethylene glycol.
步骤(3)所述的搅拌回流时间为24小时。The stirring reflux time described in step (3) is 24 hours.
所述的甘草次酸衍生物在制备甘草次酸受体介导的肝靶向脂质体中的应用。The application of the glycyrrhetinic acid derivatives in the preparation of liver targeting liposomes mediated by glycyrrhetinic acid receptors.
本发明提供的两亲性化合物——乙酰甘草次酸-聚乙二醇-硬脂酸(AGA-PEG2000-ST)位于分子链一端的甘草次酸提供了肝实质细胞靶向性，聚乙二醇片段则为脂质体提供了长循环性，疏水性的硬脂酸长烷烃链则作为锚定片段插入脂质体的脂双层中，能够赋予脂质体同时具备长循环和肝实质细胞靶向的性质，且易于插入脂质体脂双层。The amphiphilic compound provided by the present invention—acetylglycyrrhetinic acid-polyethylene glycol-stearic acid (AGA-PEG2000 -ST) glycyrrhetinic acid at one end of the molecular chain provides liver parenchymal cell targeting, polyethylene glycol The diol segment provides liposomes with long circulation, and the hydrophobic long alkane chain of stearic acid is used as an anchor segment to insert into the lipid bilayer of liposomes, which can endow liposomes with both long circulation and liver parenchyma Cell-targeting properties and easy insertion into liposome lipid bilayers.
本发明将乙酰甘草次酸-聚乙二醇-硬脂酸(AGA-PEG2000-ST)作为构成脂质体的组分，其中，乙酰甘草次酸-聚乙二醇-硬脂酸占总脂质的摩尔百分含量为0.9％～27.5％，其余组分为磷脂或磷脂衍生物、胆固醇或胆固醇衍生物以及其他可构成脂质体的两亲性化合物。本发明含有AGA-PEG2000-ST的脂质体通过通用的技术包载化合物制备肝靶向长循环脂质体载体，所包载的化合物包括临床上应用的药物、诊断试剂、影像学造影剂、生物活性物质等，可包载其中单一化合物或它们的组合物，实现单独给药或联合给药或同时给药与示踪。The present invention uses acetylglycyrrhetinic acid-polyethylene glycol-stearic acid (AGA-PEG2000 -ST) as a component of liposomes, wherein acetylglycyrrhetinic acid-polyethylene glycol-stearic acid accounts for the total The mole percentage of lipid is 0.9%-27.5%, and the rest components are phospholipids or phospholipid derivatives, cholesterol or cholesterol derivatives and other amphiphilic compounds that can constitute liposomes. The liposome containing AGA-PEG2000 -ST of the present invention prepares the liver-targeted long-circulation liposome carrier by carrying compounds in the general technology, and the carried compounds include clinically applied drugs, diagnostic reagents, imaging contrast agents , biologically active substances, etc., which can contain a single compound or their composition to achieve single administration or joint administration or simultaneous administration and tracking.
本发明具有以下优点和有益效果：The present invention has the following advantages and beneficial effects:
(1)本发明先将甘草次酸的羟基进行保护，再依次与聚乙二醇、硬脂酸进行反应，产物比较单一，操作步骤简单易行，后处理方便，有利于工业化生产；(1) The present invention first protects the hydroxyl group of glycyrrhetinic acid, and then reacts with polyethylene glycol and stearic acid in turn, the product is relatively single, the operation steps are simple and easy, and the aftertreatment is convenient, which is beneficial to industrial production;
(2)本发明所使用的硬脂酸是动植物油脂的主要成分，廉价易得，安全性很高，作为疏水长链能很轻易插入脂质体的脂双层中；而现有的技术以磷脂为插入片段，则存在着合成、分离、纯化较繁琐、稳定性低以及原料昂贵的问题；(2) stearic acid used in the present invention is the main component of animal and vegetable oils, which is cheap and easy to get, and is very safe, and can be easily inserted into the lipid bilayer of liposomes as a long hydrophobic chain; and the existing technology With phospholipids as inserts, there are problems of complicated synthesis, separation and purification, low stability and expensive raw materials;
(3)本发明所合成的化合物中，甘草次酸和硬脂酸分别位于聚乙二醇的两端，有利于甘草次酸与肝细胞表面的甘草次酸受体相互作用，提高了靶向效率；(3) In the synthesized compound of the present invention, glycyrrhetinic acid and stearic acid are respectively located at the two ends of polyethylene glycol, which is beneficial to the interaction between glycyrrhetinic acid and the glycyrrhetinic acid receptor on the surface of liver cells, and improves the targeting efficiency;
(4)本发明合成的肝靶向长循环隐形脂质体稳定性好，分散均匀集中，粒径均在100～200nm之间，能轻易躲过体内单核吞噬细胞系统(MPS)的吞噬，而到达肝实质细胞；(4) The liver-targeted long-circulation stealth liposome synthesized by the present invention has good stability, is uniformly dispersed and concentrated, and has a particle diameter between 100 and 200 nm, and can easily escape the phagocytosis of the mononuclear phagocyte system (MPS) in the body. And reach the liver parenchymal cells;
(5)采用本发明提供的甘草次酸衍生物，与常规的脂质体组分一起，采取通用的制备方法可得到包载药物或造影剂或临床试剂的脂质体，合成的脂质体作为一种纳米药物载体，既可以单独给药，也可以联合给药，可用于临床上肝脏疾病的早期诊断、治疗、示踪、药效监测等。(5) Adopting the glycyrrhetinic acid derivative provided by the present invention, together with conventional liposome components, adopting a general preparation method can obtain liposomes carrying drugs or contrast agents or clinical reagents, and the synthesized liposomes As a nano-drug carrier, it can be administered alone or in combination, and can be used for early diagnosis, treatment, tracing, and drug efficacy monitoring of liver diseases in clinical practice.
附图说明Description of drawings
图1为乙酰甘草次酸的核磁共振图谱。Figure 1 is the nuclear magnetic resonance spectrum of acetylglycyrrhetinic acid.
图2为乙酰甘草次酸-聚乙二醇2000的核磁共振图谱。Figure 2 is the nuclear magnetic resonance spectrum of acetylglycyrrhetinic acid-polyethylene glycol2000 .
图3为乙酰甘草次酸-聚乙二醇2000-硬脂酸的核磁共振图谱。Fig. 3 is the NMR spectrum of acetylglycyrrhetinic acid-polyethylene glycol2000 -stearic acid.
具体实施方式detailed description
下面结合具体实施例对本发明作进一步的描述，下列具体实施方式并非用于对本发明技术方案的限制。The present invention will be further described below in conjunction with specific examples, and the following specific embodiments are not intended to limit the technical solution of the present invention.
下述实施例中所使用的聚乙二醇为市售聚乙二醇，标号分子量为2000。The polyethylene glycol used in the following examples is commercially available polyethylene glycol with a molecular weight of 2000.
实施例1Example 1
(1)乙酰甘草次酸的合成：称取10mmol的甘草次酸(GA)置于50mL的烧瓶中，加入10ml二氯甲烷(DCM)进行溶解；然后加入10mmol的碳酸钾(K2CO3)作为缚酸剂，放入磁子，接干燥管，在冰浴条件下缓慢滴加50mmol乙酰氯(AC)，然后室温下搅拌反应8h；反应结束后，过滤除去缚酸剂；然后加入等体积的冰水，充分振摇后静置分层，收集有机层——二氯甲烷(DCM)层溶液；将有机层依次用等体积的饱和食盐水、二次蒸馏水(DDI)各洗三次，以除掉部分溶解的缚酸剂；最后加入适量的无水硫酸镁进行干燥，常压过滤除去无水硫酸镁，收集滤液，将滤液减压旋干，油泵抽2h后，放入真空干燥箱干燥，即得到乙酰甘草次酸(AGA)。图1为乙酰甘草次酸的核磁共振图谱；其中，δ(ppm)＝5.71(s,1H,-CO-CH＝C，f)处为甘草次酸多元环上的不饱和双键峰，4.50(t,1H,-CO-O-CH,a)处的峰为与酯键相邻的甘草次酸环上的氢峰,δ(ppm)＝2.350(s,1H,e)，2.816(d,1H,b)，2.072(q,1H,c)，1.832(t,3H,d)，δ(ppm)＝0.684～1.624处的峰均为甘草次酸上的特征峰。(1) Synthesis of acetylglycyrrhetinic acid: Weigh 10mmol of glycyrrhetinic acid (GA) into a 50mL flask, add 10ml of dichloromethane (DCM) to dissolve; then add 10mmol of potassium carbonate (K2 CO3 ) As an acid-binding agent, put in a magnet, connect a drying tube, slowly add 50mmol of acetyl chloride (AC) dropwise under ice bath conditions, and then stir and react at room temperature for 8h; after the reaction, remove the acid-binding agent by filtration; then add an equal volume of After fully shaking, let stand to separate layers, collect the organic layer—dichloromethane (DCM) layer solution; wash the organic layer three times with equal volumes of saturated saline and double distilled water (DDI) successively, and Remove part of the dissolved acid-binding agent; finally add an appropriate amount of anhydrous magnesium sulfate to dry, remove the anhydrous magnesium sulfate by normal pressure filtration, collect the filtrate, spin the filtrate to dry under reduced pressure, pump the oil for 2 hours, and put it in a vacuum drying oven for drying , to obtain acetylglycyrrhetinic acid (AGA). Fig. 1 is the nuclear magnetic resonance spectrum of acetylglycyrrhetinic acid; Wherein, δ (ppm)=5.71 (s, 1H,-CO-CH=C, f) place is the unsaturated double bond peak on the multiple ring of glycyrrhetinic acid, 4.50 The peak at (t, 1H, -CO-O-CH, a) is the hydrogen peak on the glycyrrhetinic acid ring adjacent to the ester bond, δ (ppm) = 2.350 (s, 1H, e), 2.816 (d ,1H,b), 2.072(q,1H,c), 1.832(t,3H,d), the peaks at δ(ppm)=0.684～1.624 are the characteristic peaks of glycyrrhetinic acid.
(2)乙酰甘草次酸-聚乙二醇的合成：取4mmol步骤(1)中合成的乙酰甘草次酸(AGA)置于50ml烧瓶中，放入磁子，加入适量的二氯甲烷溶解，制备乙酰甘草次酸的二氯甲烷溶液；将4mmol聚乙二醇(PEG2000)、4mmol二环己基碳二亚胺(DCC)、0.4mmol二甲氨基吡啶(DMAP)一同溶解在少量的二氯甲烷(DCM)中，边搅拌边缓慢滴加到乙酰甘草次酸的二氯甲烷溶液中，油浴搅拌回流24小时，反应过程中用薄层色谱监测实验进程；反应结束后，首先用少量饱和食盐水洗三次，以除掉多余的二甲氨基吡啶(DMAP)；然后加入适量无水硫酸镁干燥，过滤除去无水硫酸镁，收集滤液；滤液减压浓缩到2ml，在不断搅拌下，将浓缩液缓慢滴加到10倍体积量的冷乙醚中，放入冰箱中静置沉降24小时后，过滤，收集固体物；将固体物用少量二氯甲烷溶解后用冷乙醚沉降，重复该步骤，直至薄层层析检测无原料为止。将所得固体减压抽滤，常温油泵抽除溶剂2h后，真空干燥，即得到浅黄色粉末状乙酰甘草次酸-聚乙二醇(AGA–PEG2000)。图2为乙酰甘草次酸-聚乙二醇2000的核磁共振图谱。其中，δ(ppm)＝5.71(s,1H,-CO-CH＝C，f)，δ(ppm)＝2.350(s,1H,e)，2.816(d,1H,b)2.072(q,1H,c)，1.832(t,3H,d)处的峰均为乙酰甘草次酸上的峰，这些峰均未消失。并且在δ(ppm)＝3.65(s,192H,-O-CH2-CH2-O)处出现一个新的强峰，为聚乙二醇链上的亚甲基氢峰。δ(ppm)＝4.240(-CO-O-CH,-CO-O-CH2-a)处的峰为与酯键相邻的甘草次酸环上的氢峰和与酯键直接相连的聚乙二醇上的亚甲基的氢峰，二者部分重合。(2) Synthesis of acetylglycyrrhetinic acid-polyethylene glycol: get acetylglycyrrhetinic acid (AGA) synthesized in 4mmol step (1) and place in a 50ml flask, put into a magnet, add an appropriate amount of dichloromethane to dissolve, Prepare a dichloromethane solution of acetylglycyrrhetinic acid; dissolve 4 mmol of polyethylene glycol (PEG2000 ), 4 mmol of dicyclohexylcarbodiimide (DCC), and 0.4 mmol of dimethylaminopyridine (DMAP) in a small amount of dichloromethane In methane (DCM), slowly drop in the dichloromethane solution of acetylglycyrrhetinic acid while stirring, stir and reflux in an oil bath for 24 hours, monitor the experimental progress with thin-layer chromatography during the reaction; after the reaction finishes, first use a small amount of saturated Wash with salt water three times to remove excess dimethylaminopyridine (DMAP); then add an appropriate amount of anhydrous magnesium sulfate to dry, filter to remove anhydrous magnesium sulfate, and collect the filtrate; the filtrate is concentrated under reduced pressure to 2ml, and under constant stirring, the concentrated The solution was slowly added dropwise to 10 times the volume of cold ether, put it in the refrigerator and let it settle for 24 hours, then filtered to collect the solid matter; the solid matter was dissolved with a small amount of dichloromethane and settled with cold ether, and this step was repeated. Until there is no raw material detected by thin layer chromatography. The obtained solid was suction-filtered under reduced pressure, the solvent was removed by an oil pump at room temperature for 2 hours, and then vacuum-dried to obtain acetylglycyrrhetinic acid-polyethylene glycol (AGA-PEG2000 ) in the form of light yellow powder. Figure 2 is the nuclear magnetic resonance spectrum of acetylglycyrrhetinic acid-polyethylene glycol2000 . Among them, δ(ppm)=5.71(s,1H,-CO-CH=C,f), δ(ppm)=2.350(s,1H,e), 2.816(d,1H,b)2.072(q,1H , c), the peaks at 1.832 (t, 3H, d) are all peaks on acetylglycyrrhetinic acid, and these peaks have not disappeared. And a new strong peak appeared at δ(ppm)=3.65(s,192H,-O-CH2-CH2-O), which was the methylene hydrogen peak on the polyethylene glycol chain. The peak at δ (ppm) = 4.240 (-CO-O-CH, -CO-O-CH2-a) is the hydrogen peak on the glycyrrhetinic acid ring adjacent to the ester bond and the polyethylene glycol directly connected to the ester bond. The hydrogen peaks of the methylene groups on the diols partially overlap.
(3)硬脂酰氯的合成：称取2mmol硬脂酸置于50mL烧瓶中，加入3mL无水二氯亚砜(SOCl2)，放入磁子，接回流冷凝管、干燥管；然后将烧瓶置于磁力搅拌器上，60℃油浴搅拌回流反应4小时；反应结束后，将反应液减压旋蒸1小时后再用油泵抽气1小时，以除掉未反应完全的二氯亚砜(SOCl2)，烧瓶中剩余浅黄色固体即为制得的硬脂酰氯(STC)。(3) Synthesis of stearyl chloride: Weigh 2mmol of stearic acid and place it in a 50mL flask, add 3mL of anhydrous thionyl chloride (SOCl2 ), put in a magnet, connect a reflux condenser and a drying tube; then place the flask Put it on a magnetic stirrer, stir and reflux in an oil bath at 60°C for 4 hours; after the reaction, decompress and rotary steam the reaction solution for 1 hour, and then use an oil pump to pump air for 1 hour to remove unreacted thionyl chloride (SOCl2 ), and the light yellow solid remaining in the flask is the obtained stearyl chloride (STC).
(4)乙酰甘草次酸-聚乙二醇-硬脂酸的合成：向步骤(3)反应生成的硬脂酰氯(STC)(也可由市售硬脂酰氯替代)中加入20ml二氯甲烷(DCM)、0.02mmol二甲氨基吡啶(DMAP)、1mmol三乙胺(Et3N)；将1mmol乙酰甘草次酸-聚乙二醇(AGA–PEG2000)溶解在5ml二氯甲烷中后，冰浴条件下缓慢滴加入烧瓶，油浴搅拌回流24小时后，将反应液过滤，除掉沉淀三乙胺盐酸盐(Et3N·HCl)，然后用5ml0.1mol/L的稀盐酸洗三次，以除掉多余的三乙胺和二甲氨基吡啶，少量二次蒸馏水(DDI)洗至水相pH为中性，收集有机相；有机相溶液减压旋蒸浓缩后，边搅拌边缓慢滴加到8倍体积量的冰乙醚中，放入冰箱中静置冷藏沉降24小时后，冷过滤，收集滤出物；重复二氯甲烷溶解、乙醚沉淀三次后，减压抽滤，油泵抽干后，放入真空干燥箱干燥，即得到浅黄色粉末状的乙酰甘草次酸-聚乙二醇-硬脂酸(AGA-PEG2000-ST)。图3为乙酰甘草次酸-聚乙二醇2000-硬脂酸的核磁共振图谱；其中，δ(ppm)＝5.70ppm(s,1H,-CO-CH＝C,f)处为甘草次酸多元环上的不饱和双键峰，δ(ppm)＝4.240(-CO-O-CH,-CO-O-CH2-,a)处的峰为与酯键相邻的甘草次酸环上的氢峰和与酯键直接相连的聚乙二醇上的亚甲基的氢峰，δ(ppm)＝3.65(s,192H,-O-CH2-CH2-O)强峰，为聚乙二醇链上的亚甲基氢峰，这些特征峰都未消失。并且在δ(ppm)＝1.252(m,32H,-CH2-CH2-)处出现了强峰，是硬脂酸中亚甲基重复单元的峰，在δ(ppm)＝2.20(t,2H,-CH2-CH2-CO-O-)处的峰为与酯键相邻的硬脂酸上的氢峰，与甘草次酸酸上的e峰部分重合。(4) the synthesis of acetylglycyrrhetinic acid-polyethylene glycol-stearic acid: add 20ml methylene dichloride (STC) (also can be replaced by commercially available stearyl chloride) in the stearyl chloride (STC) that step (3) reacts to generate DCM), 0.02mmol dimethylaminopyridine (DMAP), 1mmol triethylamine (Et3 N); after dissolving 1mmol acetylglycyrrhetinic acid-polyethylene glycol (AGA–PEG2000 ) in 5ml dichloromethane, ice Slowly add it dropwise to the flask under bath conditions, stir and reflux in the oil bath for 24 hours, filter the reaction solution, remove the precipitated triethylamine hydrochloride (Et3 N HCl), and then wash it three times with 5ml of 0.1mol/L dilute hydrochloric acid , to remove excess triethylamine and dimethylaminopyridine, wash with a small amount of double distilled water (DDI) until the pH of the aqueous phase is neutral, and collect the organic phase; after the organic phase solution is concentrated by rotary evaporation under reduced pressure, slowly drop it while stirring Add it to 8 times the volume of glacial ether, put it in the refrigerator and let it settle for 24 hours, then cold filter and collect the filtrate; repeat the dissolution of dichloromethane and the precipitation of ether three times, filter under reduced pressure, and pump dry Afterwards, put it into a vacuum drying oven for drying to obtain light yellow powdered acetylglycyrrhetinic acid-polyethylene glycol-stearic acid (AGA-PEG2000 -ST). Fig. 3 is the NMR spectrum of acetylglycyrrhetinic acid-polyethylene glycol2000 -stearic acid; Wherein, δ (ppm)=5.70ppm (s, 1H,-CO-CH=C, f) place is glycyrrhetinic acid The unsaturated double bond peak on the polycyclic ring, the peak at δ (ppm) = 4.240 (-CO-O-CH, -CO-O-CH2 -, a) is on the glycyrrhetinic acid ring adjacent to the ester bond The hydrogen peak of the hydrogen peak and the hydrogen peak of the methylene on the polyethylene glycol directly connected with the ester bond, δ (ppm) = 3.65 (s, 192H, -O-CH2 -CH2 -O) strong peak, is poly The methylene hydrogen peaks on the ethylene glycol chain, none of these characteristic peaks disappeared. And at δ (ppm) = 1.252 (m, 32H, -CH2 -CH2 -) there is a strong peak, which is the peak of methylene repeating unit in stearic acid, at δ (ppm) = 2.20 (t, The peak at 2H, -CH2 -CH2 -CO-O-) is the hydrogen peak on the stearic acid adjacent to the ester bond, which partially overlaps with the e peak on the glycyrrhetinic acid.
实施例2Example 2
(1)乙酰甘草次酸的合成：称取10mmol的甘草次酸(GA)置于50mL的烧瓶中，加入10ml二氯甲烷(DCM)进行溶解；然后加入10mmol三乙胺(Et3N)作为缚酸剂，放入磁子，接干燥管，在冰浴条件下缓慢滴加60mmol的乙酰氯(AC)，然后室温搅拌反应16h；反应结束后，过滤除去缚酸剂，然后加入等体积的冰水，充分振摇后静置分层，收集有机相，有机相依次用等体积的饱和食盐水、二次蒸馏水(DDI)各洗三次，以除去部分溶解的缚酸剂，最后加入适量的无水硫酸镁进行干燥，常压过滤，收集滤液，将滤液减压旋干，油泵抽1.5h后，放入真空干燥箱干燥，即得到乙酰甘草次酸(AGA)。(1) Synthesis of acetylglycyrrhetinic acid: Weigh 10mmol of glycyrrhetinic acid (GA) and place it in a 50mL flask, add 10ml of dichloromethane (DCM) to dissolve; then add 10mmol of triethylamine (Et3 N) as Acid-binding agent, put it into a magnet, connect a drying tube, slowly add 60mmol of acetyl chloride (AC) dropwise under ice bath conditions, and then stir and react at room temperature for 16h; after the reaction, remove the acid-binding agent by filtration, and then add an equal volume of Ice water, fully shaken and let stand to separate layers, collect the organic phase, and wash the organic phase with equal volume of saturated saline and double distilled water (DDI) three times respectively to remove part of the dissolved acid-binding agent, and finally add an appropriate amount of Dry over anhydrous magnesium sulfate, filter under normal pressure, collect the filtrate, spin dry the filtrate under reduced pressure, pump it with an oil pump for 1.5 hours, and put it into a vacuum drying oven for drying to obtain acetylglycyrrhetinic acid (AGA).
(2)乙酰甘草次酸-聚乙二醇的合成：取4mmol步骤(1)中合成的乙酰甘草次酸(AGA)于50ml烧瓶中，放入磁子，加入适量的二氯甲烷进行溶解，制备乙酰甘草次酸的二氯甲烷溶液；将5mmol的聚乙二醇(PEG2000)、4.8mmol二环己基碳二亚胺(DCC)、0.08mmol二甲氨基吡啶(DMAP)溶解在二氯甲烷(DCM)中，边搅拌边缓慢滴加到乙酰甘草次酸的二氯甲烷溶液中，油浴搅拌回流18小时，反应过程中不断用薄层色谱监测实验进程；反应结束后，首先用少量饱和食盐水洗三次，以除掉多余的二甲氨基吡啶(DMAP)，然后加入适量无水硫酸镁进行干燥，过滤除去无水硫酸镁，收集滤液，滤液减压浓缩到1.5ml，在不断搅拌的情况下，缓慢滴加到8倍体积量的冷乙醚中，放入冰箱中静置冷藏沉降24小时后，冷过滤，收集固体物。重复少量二氯甲烷溶解、乙醚沉降，直到薄层层析检测无原料点为止；将所得固体减压抽干，油泵抽2h后，真空干燥，即得到浅黄色粉末状乙酰甘草次酸-聚乙二醇(AGA–PEG2000)。(2) Synthesis of acetylglycyrrhetinic acid-polyethylene glycol: get acetylglycyrrhetinic acid (AGA) synthesized in 4mmol step (1) in a 50ml flask, put into a magnet, add an appropriate amount of dichloromethane to dissolve, Prepare a dichloromethane solution of acetylglycyrrhetinic acid; dissolve 5 mmol of polyethylene glycol (PEG2000 ), 4.8 mmol of dicyclohexylcarbodiimide (DCC), and 0.08 mmol of dimethylaminopyridine (DMAP) in dichloromethane (DCM), slowly added dropwise to the dichloromethane solution of acetylglycyrrhetinic acid while stirring, stirred and refluxed in an oil bath for 18 hours, and constantly monitored the experimental process with thin-layer chromatography in the reaction process; Wash with salt water three times to remove excess dimethylaminopyridine (DMAP), then add an appropriate amount of anhydrous magnesium sulfate to dry, filter to remove anhydrous magnesium sulfate, collect the filtrate, and concentrate the filtrate to 1.5ml under reduced pressure. Slowly add it dropwise to 8 times the volume of cold diethyl ether, put it in the refrigerator and let it settle for 24 hours, then cold filter and collect the solid. Repeat the dissolution of a small amount of dichloromethane and the precipitation of ether until there is no raw material point detected by thin-layer chromatography; the resulting solid is vacuum-dried, pumped for 2 hours, and then vacuum-dried to obtain light yellow powder acetylglycyrrhetinic acid-polyethylene Diol (AGA–PEG2000 ).
(3)硬脂酰氯的合成：称取1mmol硬脂酸置于50mL烧瓶中，加入3mL无水二氯亚砜(SOCl2)，放入磁子，接回流冷凝管、干燥管。置于磁力搅拌器上，60℃油浴搅拌回流反应4小时。停止反应，将反应液减压旋蒸1小时后再用油泵抽气1小时，以除掉未反应完全的二氯亚砜(SOCl2)，烧瓶中剩余浅黄色固体即为制得的硬脂酰氯(STC)。(3) Synthesis of stearyl chloride: Weigh 1 mmol of stearic acid and put it in a 50 mL flask, add 3 mL of anhydrous thionyl chloride (SOCl2 ), put in a magnet, connect a reflux condenser tube and a drying tube. Place on a magnetic stirrer, stir and reflux in an oil bath at 60°C for 4 hours. Stop the reaction, depressurize the reaction solution for 1 hour and pump it with an oil pump for 1 hour to remove unreacted thionyl chloride (SOCl2 ), and the remaining light yellow solid in the flask is the obtained stearin acid chloride (STC).
(4)乙酰甘草次酸-聚乙二醇-硬脂酸的合成：向步骤(3)反应生成的硬脂酰氯(STC)中加入20ml二氯甲烷(DCM)、0.1mmol二甲氨基吡啶(DMAP)、1mmol三乙胺(Et3N)；将1mmol乙酰甘草次酸-聚乙二醇(AGA–PEG2000)溶解在5ml二氯甲烷中，冰浴条件下缓慢滴加入烧瓶，油浴搅拌回流30小时后，将反应液过滤，除掉沉淀三乙胺盐酸盐(Et3N.HCl)，然后用5ml0.1mol/L的稀盐酸洗三次，以除掉多余的三乙胺和二甲氨基吡啶，少量二次蒸馏水(DDI)洗至水相pH为中性，收集有机相；有机相溶液减压旋蒸浓缩后，边搅拌边缓慢滴加到10倍体积量的冰乙醚中，放入冰箱中静置冷藏沉降24小时后，冷过滤，收集滤出物；重复二氯甲烷溶解、乙醚沉淀三次后，减压抽滤，油泵抽干后，放入真空干燥箱干燥，即得到浅黄色粉末状的乙酰甘草次酸-聚乙二醇-硬脂酸(AGA-PEG2000-ST)。(4) the synthesis of acetylglycyrrhetinic acid-polyethylene glycol-stearic acid: add 20ml dichloromethane (DCM), 0.1mmol dimethylaminopyridine ( DMAP), 1mmol triethylamine (Et3 N); 1mmol acetylglycyrrhetinic acid-polyethylene glycol (AGA–PEG2000 ) was dissolved in 5ml dichloromethane, slowly added dropwise to the flask under ice-bath conditions, and stirred in an oil bath After refluxing for 30 hours, the reaction solution was filtered to remove precipitated triethylamine hydrochloride (Et3 N.HCl), and then washed three times with 5ml of 0.1mol/L dilute hydrochloric acid to remove excess triethylamine and diethylamine. Aminopyridine, washed with a small amount of double distilled water (DDI) until the pH of the aqueous phase is neutral, and the organic phase is collected; after the organic phase solution is concentrated by rotary evaporation under reduced pressure, it is slowly added dropwise to 10 times the volume of glacial ether while stirring, Put it in the refrigerator for 24 hours, then cold filter and collect the filtrate; after repeating dichloromethane dissolution and ether precipitation three times, filter under reduced pressure, drain the oil pump, and dry in a vacuum oven to obtain Acetylglycyrrhetinic acid-polyethylene glycol-stearic acid (AGA-PEG2000 -ST) in pale yellow powder form.
实施例3Example 3
(1)乙酰甘草次酸的合成：称取10mmol的甘草次酸(GA)溶解在10ml二氯甲烷(DCM)于50mL烧瓶中，加入20mmol的碳酸钾(K2CO3)作为缚酸剂，放入磁子，接干燥管，在冰浴条件下缓慢滴加100mmol的乙酰氯(AC)，然后室温搅拌反应24h；反应结束后，过滤除去缚酸剂，然后加入等体积的冰水，充分振摇后静置分层，收集二氯甲烷(DCM)层溶液，并依次用等体积的饱和食盐水、二次蒸馏水(DDI)各洗三次，以除去部分溶解的碳酸钾，最后加入适量的无水硫酸镁进行干燥，常压过滤，收集滤液，将滤液减压旋干，油泵抽1h后，放入真空干燥箱干燥，得到乙酰甘草次酸(AGA)。(1) Synthesis of acetylglycyrrhetinic acid: Weigh 10mmol of glycyrrhetinic acid (GA) and dissolve it in 10ml of dichloromethane (DCM) in a 50mL flask, add 20mmol of potassium carbonate (K2 CO3 ) as an acid-binding agent, Put in a magnet, connect a drying tube, slowly add 100 mmol of acetyl chloride (AC) dropwise under ice bath conditions, and then stir at room temperature for 24 hours; after the reaction, remove the acid-binding agent by filtration, then add an equal volume of ice water, fully After shaking, let it stand for stratification, collect the dichloromethane (DCM) layer solution, and wash it three times with an equal volume of saturated saline and double distilled water (DDI) successively to remove part of the dissolved potassium carbonate, and finally add an appropriate amount of Dry over anhydrous magnesium sulfate, filter under normal pressure, collect the filtrate, spin dry the filtrate under reduced pressure, pump it with an oil pump for 1 hour, and put it into a vacuum drying oven to dry to obtain acetylglycyrrhetinic acid (AGA).
(2)乙酰甘草次酸-聚乙二醇的合成：取4mmol步骤(1)中合成的乙酰甘草次酸(AGA)于50ml烧瓶中，放入磁子，加入20ml二氯甲烷溶解，制备乙酰甘草次酸的二氯甲烷溶液；将4.5mmol的聚乙二醇(PEG2000)、4.5mmol二环己基碳二亚胺(DCC)、0.1mmol二甲氨基吡啶(DMAP)一同溶解在二氯甲烷中，边搅拌边缓慢滴加到乙酰甘草次酸的二氯甲烷溶液中，油浴搅拌回流30小时，反应过程中不断用薄层色谱监测实验进程；反应结束后，首先用少量饱和食盐水洗三次，以除掉多余的二甲氨基吡啶(DMAP)，然后加入适量无水硫酸镁进行干燥，然后过滤除去无水硫酸镁，收集滤液，将滤液减压浓缩到1ml；然后在不断搅拌的条件下，将滤液浓缩液缓慢滴加到10倍体积量的冷乙醚中，放入冰箱中静置冷藏沉降24小时后，冷过滤，收集固体物；重复少量二氯甲烷溶解、乙醚沉降，直到薄层层析检测无原料点为止；将所得固体减压抽干，油泵抽2h后，真空干燥，即得到浅黄色粉末状乙酰甘草次酸-聚乙二醇(AGA–PEG2000)。(2) Synthesis of acetylglycyrrhetinic acid-polyethylene glycol: get acetylglycyrrhetinic acid (AGA) synthesized in 4mmol step (1) in a 50ml flask, put into a magnet, add 20ml of dichloromethane to dissolve, and prepare acetylglycyrrhetinic acid (AGA) Dichloromethane solution of glycyrrhetinic acid; 4.5mmol of polyethylene glycol (PEG2000 ), 4.5mmol of dicyclohexylcarbodiimide (DCC), and 0.1mmol of dimethylaminopyridine (DMAP) were dissolved together in dichloromethane in the dichloromethane solution of acetylglycyrrhetinic acid slowly while stirring, the oil bath was stirred and refluxed for 30 hours, and the experimental progress was continuously monitored by thin-layer chromatography during the reaction process; , to remove excess dimethylaminopyridine (DMAP), then add an appropriate amount of anhydrous magnesium sulfate to dry, then filter to remove anhydrous magnesium sulfate, collect the filtrate, and concentrate the filtrate to 1ml under reduced pressure; then under constant stirring , slowly add the filtrate concentrate dropwise to 10 times the volume of cold ether, put it in the refrigerator and let it stand for 24 hours to refrigerate and settle, then cold filter to collect the solid matter; repeat a small amount of dichloromethane to dissolve and ether to settle until the thin layer Chromatographic detection until there is no raw material point; the obtained solid is vacuum-dried, oil pumped for 2 hours, and vacuum-dried to obtain light yellow powder acetylglycyrrhetinic acid-polyethylene glycol (AGA-PEG2000 ).
(3)乙酰甘草次酸-聚乙二醇-硬脂酸的合成：向2mmol硬脂酰氯(STC)(市售，阿拉丁)中加入20ml二氯甲烷(DCM)、0.08mmol二甲氨基吡啶(DMAP)、2mmol三乙胺(Et3N)；将1mmol乙酰甘草次酸-聚乙二醇(AGA–PEG2000)溶解在5ml二氯甲烷中，冰浴条件下将其缓慢滴入烧瓶中，油浴搅拌回流18小时后，将反应液过滤，除掉沉淀三乙胺盐酸盐(Et3N.HCl)，然后用5ml0.1mol/L的稀盐酸洗三次，以除掉多余的三乙胺和二甲氨基吡啶，少量二次蒸馏水(DDI)洗至水相pH为中性，收集有机相。有机相溶液减压旋蒸浓缩后，边搅拌边缓慢滴加到6倍体积量的冰乙醚中，放入冰箱中静置冷藏沉降24小时后，冷过滤，收集滤出物；重复二氯甲烷溶解、乙醚沉淀三次后，减压抽滤，油泵抽干后，放入真空干燥箱干燥。得到浅黄色粉末，即为乙酰甘草次酸-聚乙二醇-硬脂酸(AGA-PEG2000-ST)。(3) Synthesis of acetylglycyrrhetinic acid-polyethylene glycol-stearic acid: add 20ml dichloromethane (DCM), 0.08mmol dimethylaminopyridine to 2mmol stearyl chloride (STC) (commercially available, Aladdin) (DMAP), 2mmol triethylamine (Et3 N); 1mmol acetylglycyrrhetinic acid-polyethylene glycol (AGA–PEG2000 ) was dissolved in 5ml dichloromethane, and slowly dropped into the flask under ice-cooling conditions After stirring and refluxing in an oil bath for 18 hours, the reaction solution was filtered to remove precipitated triethylamine hydrochloride (Et3 N.HCl), and then washed three times with 5ml of 0.1mol/L dilute hydrochloric acid to remove excess triethylamine hydrochloride (Et 3 N.HCl). Ethylamine and dimethylaminopyridine were washed with a small amount of double distilled water (DDI) until the pH of the aqueous phase was neutral, and the organic phase was collected. After the organic phase solution is concentrated by rotary evaporation under reduced pressure, it is slowly added dropwise to 6 times the volume of glacial ether while stirring, put it in the refrigerator and let it refrigerate and settle for 24 hours, then cold filter and collect the filtrate; repeat dichloromethane After three times of dissolution and ether precipitation, filter under reduced pressure, drain with an oil pump, and dry in a vacuum oven. The light yellow powder obtained is acetylglycyrrhetinic acid-polyethylene glycol-stearic acid (AGA-PEG2000 -ST).
实施例4Example 4
(1)乙酰甘草次酸的合成：称取10mmol的甘草次酸(GA)溶解在10ml二氯甲烷(DCM)于50mL烧瓶中，加入15mmol三乙胺(Et3N)作为缚酸剂，放入磁子，接干燥管，在冰浴条件下缓慢滴加80mmol的乙酰氯(AC)，然后室温搅拌反应16h；反应结束后，过滤除去缚酸剂，然后加入等体积的冰水，充分振摇后静置分层，收集二氯甲烷(DCM)层溶液，并依次用等体积的饱和食盐水、二次蒸馏水(DDI)各洗三次，以除去部分溶解缚酸剂，最后加入适量的无水硫酸镁干燥，常压过滤，收集滤液，将滤液减压旋干，油泵抽1.5h后，放入真空干燥箱干燥，即得到乙酰甘草次酸(AGA)。(1) Synthesis of acetylglycyrrhetinic acid: take 10mmol of glycyrrhetinic acid (GA) and dissolve it in 10ml of dichloromethane (DCM) in a 50mL flask, add 15mmol of triethylamine (Et3 N) as an acid-binding agent, put into the magneton, connected to a drying tube, and slowly added 80 mmol of acetyl chloride (AC) dropwise under ice bath conditions, then stirred at room temperature for 16 hours; After shaking, let it stand for stratification, collect the dichloromethane (DCM) layer solution, and wash it three times with an equal volume of saturated saline and double distilled water (DDI) successively to remove part of the dissolved acid-binding agent, and finally add an appropriate amount of Dry over magnesium sulfate, filter under normal pressure, collect the filtrate, spin dry the filtrate under reduced pressure, pump it with an oil pump for 1.5 hours, and put it in a vacuum drying oven to dry to obtain acetylglycyrrhetinic acid (AGA).
(2)乙酰甘草次酸-聚乙二醇的合成：取4mmol步骤(1)中合成的乙酰甘草次酸(AGA)于50ml烧瓶中，放入磁子，加入适量的二氯甲烷进行溶解，制备乙酰甘草次酸的二氯甲烷溶液；将4.3mmol的聚乙二醇(PEG2000)、4.6mmol二环己基碳二亚胺(DCC)、0.2mmol二甲氨基吡啶(DMAP)溶解在二氯甲烷(DCM)中，边搅拌边缓慢滴加到乙酰甘草次酸的二氯甲烷溶液中，油浴搅拌回流24小时，反应过程中不断用薄层色谱监测实验进程；反应结束后，首先用少量饱和食盐水洗三次，以除掉多余的二甲氨基吡啶(DMAP)，然后加入适量无水硫酸镁进行干燥，过滤除去无水硫酸镁，收集滤液，滤液减压浓缩到1.5ml，在不断搅拌的情况下，缓慢滴加到8倍体积量的冷乙醚中，放入冰箱中静置冷藏沉降24小时后，冷过滤，收集固体物。重复少量二氯甲烷溶解、乙醚沉降，直到薄层层析检测无原料点为止；将所得固体减压抽干，油泵抽2h后，真空干燥，即得到浅黄色粉末状乙酰甘草次酸-聚乙二醇(AGA–PEG2000)。(2) Synthesis of acetylglycyrrhetinic acid-polyethylene glycol: get acetylglycyrrhetinic acid (AGA) synthesized in 4mmol step (1) in a 50ml flask, put into a magnet, add an appropriate amount of dichloromethane to dissolve, Prepare a dichloromethane solution of acetylglycyrrhetinic acid; dissolve 4.3 mmol of polyethylene glycol (PEG2000 ), 4.6 mmol of dicyclohexylcarbodiimide (DCC), and 0.2 mmol of dimethylaminopyridine (DMAP) in dichloromethane In methane (DCM), slowly drop in the dichloromethane solution of acetylglycyrrhetinic acid while stirring, stir and reflux in an oil bath for 24 hours, constantly monitor the experimental process with thin-layer chromatography in the reaction process; after the reaction finishes, first use a small amount of Wash with saturated brine three times to remove excess dimethylaminopyridine (DMAP), then add an appropriate amount of anhydrous magnesium sulfate to dry, filter to remove anhydrous magnesium sulfate, collect the filtrate, and concentrate the filtrate under reduced pressure to 1.5ml. Under the circumstances, slowly add it dropwise to 8 times the volume of cold ether, put it in the refrigerator and let it settle for 24 hours, then cold filter and collect the solid. Repeat the dissolution of a small amount of dichloromethane and the precipitation of ether until there is no raw material point detected by thin-layer chromatography; the resulting solid is vacuum-dried, pumped for 2 hours, and then vacuum-dried to obtain light yellow powder acetylglycyrrhetinic acid-polyethylene Diol (AGA–PEG2000 ).
(3)乙酰甘草次酸-聚乙二醇-硬脂酸的合成：向2mmol硬脂酰氯(STC，阿拉丁)中加入20ml二氯甲烷(DCM)、0.05mmol二甲氨基吡啶(DMAP)、1.5mmol三乙胺(Et3N)；将1mmol乙酰甘草次酸-聚乙二醇(AGA–PEG2000)溶解在5ml二氯甲烷中，冰浴条件下缓慢滴加入烧瓶，油浴搅拌回流30小时后，将反应液过滤，除掉沉淀三乙胺盐酸盐(Et3N.HCl)，然后用5ml0.1mol/L的稀盐酸洗三次，以除掉多余的三乙胺和二甲氨基吡啶，少量二次蒸馏水(DDI)洗至水相pH为中性，收集有机相；有机相溶液减压旋蒸浓缩后，边搅拌边缓慢滴加到10倍体积量的冰乙醚中，放入冰箱中静置冷藏沉降24小时后，冷过滤，收集滤出物；重复二氯甲烷溶解、乙醚沉淀三次后，减压抽滤，油泵抽干后，放入真空干燥箱干燥，即得到浅黄色粉末状的乙酰甘草次酸-聚乙二醇-硬脂酸(AGA-PEG2000-ST)。(3) Synthesis of acetylglycyrrhetinic acid-polyethylene glycol-stearic acid: in 2mmol stearoyl chloride (STC, Aladdin), add 20ml dichloromethane (DCM), 0.05mmol dimethylaminopyridine (DMAP), 1.5mmol triethylamine (Et3 N); 1mmol acetylglycyrrhetinic acid-polyethylene glycol (AGA–PEG2000 ) was dissolved in 5ml dichloromethane, slowly added dropwise to the flask under ice-bath conditions, stirred and refluxed in an oil bath for 30 After 1 hour, the reaction solution was filtered to remove precipitated triethylamine hydrochloride (Et3 N.HCl), and then washed three times with 5ml of 0.1mol/L dilute hydrochloric acid to remove excess triethylamine and dimethylamino Pyridine and a small amount of double-distilled water (DDI) were washed until the pH of the aqueous phase was neutral, and the organic phase was collected; after the organic phase solution was concentrated by rotary evaporation under reduced pressure, it was slowly added dropwise to 10 times the volume of glacial ether while stirring, and put into After refrigerating and settling for 24 hours in the refrigerator, cold filter and collect the filtrate; after repeating dichloromethane dissolution and ether precipitation three times, reduce pressure and filter, drain with an oil pump, put it in a vacuum drying oven to dry, and obtain light yellow Acetylglycyrrhetinic acid-polyethylene glycol-stearic acid (AGA-PEG2000 -ST) in powder form.
实施例5：传统脂质体的制备(对照试验)Embodiment 5: the preparation of traditional liposome (control test)
称取卵磷脂(PC)20mg和胆固醇(CHO)2mg，用2ml氯仿和无水乙醇的混合溶液(1：1)溶解在250ml茄形瓶中，将溶液以每分钟40转的速度在35℃水浴真空旋转蒸干，并在40℃下真空干燥6小时以除去残留溶剂，得到均一分散的脂质薄膜；向其中加入5mL空白Tris-HCl缓冲液(pH8.8)，并在55℃水浴中，以每分钟120转的速度常压旋转水合2h后，将所得脂质体液通过高压挤出器用氮气挤出，在此过程中，多室脂质体混悬液依次经过一系列孔径为800nm、450nm、220nm的滤膜分别挤出10次后，粒径大小分布变得更均一集中。得到的脂质体的粒径为161.2nm～165.6nm，粒径分布蛋白质分散指数(PDI)为0.219～0.224。Weigh 20mg of lecithin (PC) and 2mg of cholesterol (CHO), dissolve it in a 250ml eggplant-shaped bottle with 2ml of a mixed solution of chloroform and absolute ethanol (1:1), and put the solution at 35°C at a speed of 40 rpm Rotate to dryness in a water bath, and vacuum dry at 40°C for 6 hours to remove residual solvents to obtain a uniformly dispersed lipid film; add 5mL blank Tris-HCl buffer solution (pH8. After rotating and hydrating under normal pressure at a speed of 120 revolutions per minute for 2 hours, the obtained liposome liquid is extruded with nitrogen through a high-pressure extruder. After the 450nm and 220nm filter membranes were extruded 10 times respectively, the particle size distribution became more uniform and concentrated. The particle size of the obtained liposome is 161.2nm-165.6nm, and the particle size distribution protein dispersion index (PDI) is 0.219-0.224.
实施例6：含有乙酰甘草次酸-聚乙二醇-硬脂酸的肝靶向长循环脂质体的制备Example 6: Preparation of liver-targeted long-circulation liposomes containing acetylglycyrrhetinic acid-polyethylene glycol-stearic acid
(1)称取卵磷脂(PC)20mg、胆固醇(CHO)2mg和乙酰甘草次酸-聚乙二醇-硬脂酸7.2mg，用2ml氯仿和无水乙醇的混合溶液(1：1)溶解在250ml茄形瓶中，将溶液以每分钟40转的速度在35℃真空旋转蒸发以得到均匀分散的脂质体薄膜；然后向其中加入5mL空白Tris-HCl缓冲液(pH8.8)，并在55℃水浴中，以每分钟120转的速度常压旋转水合1h～2h后，将所得脂质体液通过高压挤出器用氮气压滤，在此过程中，多室脂质体混悬液依次经过一系列孔径为800nm、450nm、220nm的滤膜分别挤出10次后，粒径大小分布变得更均一集中。得到的脂质体的粒径为137.4nm～139.9nm，粒径分布PDI为0.095～0.133。(1) Weigh 20 mg of lecithin (PC), 2 mg of cholesterol (CHO) and 7.2 mg of acetylglycyrrhetinic acid-polyethylene glycol-stearic acid, and dissolve them in a mixed solution of 2 ml of chloroform and absolute ethanol (1:1) In a 250ml eggplant-shaped bottle, the solution was rotated in vacuum at 35°C to obtain a uniformly dispersed liposome film at a speed of 40 revolutions per minute; then 5mL blank Tris-HCl buffer (pH8.8) was added thereto, and In a water bath at 55°C, rotate and hydrate under normal pressure at a speed of 120 revolutions per minute for 1h to 2h, and filter the obtained liposome liquid through a high-pressure extruder with nitrogen pressure. During this process, the multilocular liposome suspension is sequentially After a series of filter membranes with pore sizes of 800nm, 450nm, and 220nm were extruded 10 times, the particle size distribution became more uniform and concentrated. The particle size of the obtained liposome is 137.4nm-139.9nm, and the particle size distribution PDI is 0.095-0.133.
(2)称取卵磷脂(PC)20mg、胆固醇(CHO)2mg和乙酰甘草次酸-聚乙二醇-硬脂酸0.72mg，用2ml氯仿和无水乙醇的混合溶液(1：1)溶解在250ml茄形瓶中，将溶液以每分钟40转的速度在35℃真空旋转蒸发以得到均匀分散的脂质体薄膜；然后向其中加入5mL空白Tris-HCl缓冲液(pH8.8)，并在55℃水浴中，以每分钟120转的速度常压旋转水合1h～2h后，将所得脂质体液通过高压挤出器用氮气压滤，在此过程中，多室脂质体混悬液经过一系列孔径为800nm、450nm、220nm的滤膜分别压滤10次后，粒径大小分布变得更均一集中。得到的脂质体的粒径为156.2nm～161.6nm，粒径分布PDI为0.209～0.214。(2) Weigh 20 mg of lecithin (PC), 2 mg of cholesterol (CHO) and 0.72 mg of acetylglycyrrhetinic acid-polyethylene glycol-stearic acid, and dissolve them in a mixed solution of 2 ml of chloroform and absolute ethanol (1:1) In a 250ml eggplant-shaped bottle, the solution was rotated in vacuum at 35°C to obtain a uniformly dispersed liposome film at a speed of 40 revolutions per minute; then 5mL blank Tris-HCl buffer (pH8.8) was added thereto, and In a water bath at 55°C, rotate and hydrate under normal pressure at a speed of 120 revolutions per minute for 1h to 2h, then pass the obtained liposome liquid through a high-pressure extruder and filter it with nitrogen pressure. During this process, the multilocular liposome suspension passes through A series of filter membranes with pore sizes of 800nm, 450nm, and 220nm were press-filtered 10 times respectively, and the particle size distribution became more uniform and concentrated. The particle size of the obtained liposome is 156.2nm-161.6nm, and the particle size distribution PDI is 0.209-0.214.
(3)称取卵磷脂(PC)20mg和胆固醇(CHO)2mg、乙酰甘草次酸-聚乙二醇-硬脂酸21.6mg，用2ml氯仿和无水乙醇的混合溶液(1：1)溶解在250ml茄形瓶中，将溶液以每分钟40转的速度在35℃真空旋转蒸发以得到均匀分散的脂质体薄膜；然后向其中加入5mL空白Tris-HCl缓冲液(pH8.8)，并在55℃水浴中，以每分钟120转的速度常压旋转水合1h～2h后，将所得脂质体液通过高压挤出器用氮气压滤，在此过程中，多室脂质体混悬液依次经过一系列孔径为800nm、450nm、220nm的滤膜分别挤出10次后，粒径大小分布变得更均一集中。得到的脂质体的粒径为170.3nm～173.6nm，粒径分布PDI为0.210～0.223。(3) Weigh 20mg of lecithin (PC), 2mg of cholesterol (CHO), 21.6mg of acetylglycyrrhetinic acid-polyethylene glycol-stearic acid, and dissolve them in a mixed solution of 2ml of chloroform and absolute ethanol (1:1) In a 250ml eggplant-shaped bottle, the solution was rotated in vacuum at 35°C to obtain a uniformly dispersed liposome film at a speed of 40 revolutions per minute; then 5mL blank Tris-HCl buffer (pH8.8) was added thereto, and In a water bath at 55°C, rotate and hydrate under normal pressure at a speed of 120 revolutions per minute for 1h to 2h, and filter the obtained liposome liquid through a high-pressure extruder with nitrogen pressure. During this process, the multilocular liposome suspension is sequentially After a series of filter membranes with pore sizes of 800nm, 450nm, and 220nm were extruded 10 times, the particle size distribution became more uniform and concentrated. The particle size of the obtained liposome is 170.3nm-173.6nm, and the particle size distribution PDI is 0.210-0.223.
实施例7：长循环阴离子脂质体制备(对照试验)Embodiment 7: preparation of long circulation anionic liposome (control test)
为了更好地包载阳离子亲水药物，如顺铂、基因、蛋白质等带有正电荷的大分子，本发明还引入了5-胆甾烯-3β-琥珀酸单酯(CHO-HS)。In order to better pack cationic hydrophilic drugs, such as cisplatin, genes, proteins and other positively charged macromolecules, the present invention also introduces 5-cholestene-3β-succinic acid monoester (CHO-HS).
具体制法如下：称取硬脂酸-聚乙二醇单甲醚5.93mg、卵磷脂(PC)20mg和5-胆甾烯-3β-琥珀酸单酯2.5mg，用氯仿和无水乙醇的混合液(1：1)溶解在250ml茄形瓶中，将溶液以每分钟40转的速度在35℃真空旋转蒸发以得到均匀分散的脂质体薄膜；然后向其中加入5mLTris-HCl缓冲液(pH8.8)，并在55℃水浴中，以每分钟120转的速度常压旋转充分水合，将所得脂质体液体通过高压挤出器用氮气挤出，在此过程中，多室脂质体混悬液经过一系列孔径为800nm、450nm、220nm的滤膜分别挤出10次后，粒径大小分布变得更均一集中。得到的脂质体的粒径为164.8nm～167.1nm，粒径分布PDI为0.179～0.186。The specific preparation method is as follows: Weigh 5.93 mg of stearic acid-polyethylene glycol monomethyl ether, 20 mg of lecithin (PC) and 2.5 mg of 5-cholestene-3β-succinic acid monoester, and mix with chloroform and absolute ethanol The mixed solution (1:1) was dissolved in a 250ml eggplant-shaped bottle, and the solution was rotated in vacuum at 35° C. to obtain a uniformly dispersed liposome film at a speed of 40 revolutions per minute; then 5mL Tris-HCl buffer solution was added thereto ( pH8.8), and in a water bath at 55°C, fully hydrated with normal pressure rotation at a speed of 120 revolutions per minute, and the resulting liposome liquid is extruded with nitrogen through a high-pressure extruder. In the process, the multilamellar liposome After the suspension was extruded 10 times through a series of filter membranes with pore sizes of 800nm, 450nm and 220nm, the particle size distribution became more uniform and concentrated. The particle size of the obtained liposome is 164.8nm-167.1nm, and the particle size distribution PDI is 0.179-0.186.
实施例8：含有乙酰甘草次酸-聚乙二醇-硬脂酸的肝靶向长循环阴离子脂质体制备Example 8: Preparation of liver-targeted long-circulation anionic liposomes containing acetylglycyrrhetinic acid-polyethylene glycol-stearic acid
称取甘草次酸-聚乙二醇-硬脂酸7.2mg、磷脂20mg、5-胆甾烯-3β-琥珀酸单酯2.5mg，用氯仿和无水乙醇的混合液(1：1)溶解在250ml茄形瓶中，将溶液以每分钟40转的速度在35℃真空旋转蒸发以得到均匀分散的脂质薄膜；然后向其中加入5mLTris-HCl缓冲液(pH8.8)，并在55℃水浴中，以每分钟120转的速度常压旋转充分水合，将所得脂质体液体通过高压挤出器用氮气挤出，在此过程中，多室脂质体混悬液依次经过一系列孔径为800nm、450nm、220nm的滤膜分别挤出10次后，粒径大小分布变得更均一集中。得到的脂质体的粒径为147.9nm～149.3nm，粒径分布PDI为0.156～0.164。Weigh 7.2 mg of glycyrrhetinic acid-polyethylene glycol-stearic acid, 20 mg of phospholipids, and 2.5 mg of 5-cholestene-3β-succinic acid monoester, and dissolve them with a mixture of chloroform and absolute ethanol (1:1) In a 250ml eggplant-shaped flask, the solution was rotated in vacuum at 35°C at a speed of 40 rpm to obtain a uniformly dispersed lipid film; In a water bath, rotate at a speed of 120 revolutions per minute under normal pressure to fully hydrate, and the resulting liposome liquid is extruded with nitrogen through a high-pressure extruder. After the 800nm, 450nm, and 220nm filter membranes were extruded 10 times, the particle size distribution became more uniform and concentrated. The particle size of the obtained liposome is 147.9nm-149.3nm, and the particle size distribution PDI is 0.156-0.164.
实施例9Example 9
为了研究含有乙酰甘草次酸-聚乙二醇-硬脂酸脂质体的肝靶向性，将不含有乙酰甘草次酸-聚乙二醇-硬脂酸的脂质体(记作mPEG-STlipos)和含有乙酰甘草次酸-聚乙二醇-硬脂酸的脂质体(记作AGA-PEG2000-STlipos)分别包载模型药物——钙黄绿素(以游离的钙黄绿素溶液为对照)，然后将它们分别与HepG2细胞和A549细胞共同孵育8h，钙黄绿素的最终浓度为2μg/mL。然后用PBS(0.01M，pH＝7.4)缓冲液漂洗细胞3次，以除去未被细胞摄取的钙黄绿素脂质体；用4％多聚甲醛溶液在室温下避光固定15min后，用PBS缓冲液漂洗3次。用Hoechst33258(5μg/mL)在37℃下对细胞核染色10min后用PBS缓冲液再漂洗3次。通过共聚焦荧光显微镜对处理好的共聚焦培养皿进行观察。结果显示，经载有钙黄绿素的AGA-PEG2000-STlipos作用后的HepG2细胞的荧光强度明显强于载有钙黄绿素的mPEG-STlipos作用后的HepG2细胞的荧光强度；而对于A549细胞，二者的荧光强度没有明显差别。实施例10In order to study the liver targeting of liposomes containing acetylglycyrrhetinic acid-polyethylene glycol-stearate, liposomes not containing acetylglycyrrhetinic acid-polyethylene glycol-stearate (denoted as mPEG- STlipos) and liposomes (referred to as AGA-PEG 2000-STlipos) containing acetylglycyrrhetinic acid-polyethylene glycol-stearic acid (referred to as AGA-PEG2000 -STlipos) respectively entrapped the model drug-calcein (with free calcein solution as the control) , and then they were co-incubated with HepG2 cells and A549 cells for 8h, and the final concentration of calcein was 2 μg/mL. Then rinse the cells with PBS (0.01M, pH=7.4) buffer solution for 3 times to remove the calcein liposomes that were not taken up by the cells; fix with 4% paraformaldehyde solution at room temperature in the dark for 15min, then buffer with PBS Liquid rinse 3 times. Cell nuclei were stained with Hoechst33258 (5 μg/mL) at 37°C for 10 min, and then rinsed three times with PBS buffer. Observe the processed confocal culture dish by confocal fluorescence microscope. The results showed that the fluorescence intensity of HepG2 cells loaded with calcein-loaded AGA-PEG2000 -STlipos was significantly stronger than that of HepG2 cells loaded with calcein-loaded mPEG-STlipos; and for A549 cells, both There was no significant difference in fluorescence intensity. Example 10
为了更进一步的研究细胞对包载有钙黄绿素脂质体的内吞作用，将不含有乙酰甘草次酸-聚乙二醇-硬脂酸的脂质体(记作mPEG-STlipos)和含有乙酰甘草次酸-聚乙二醇-硬脂酸的脂质体(记作AGA-PEG2000-STlipos)分别包载模型药物——钙黄绿素，将它们分别与HepG2细胞和A549细胞共同孵育8h(空白细胞作为对照)，钙黄绿素的最终浓度为2μg/mL。然后吸出培养液，用PBS(0.01M，pH＝7.4)缓冲液清洗培养板三遍后，加入胰蛋白酶消化使细胞脱壁，用不含血清的培养基终止消化，消化液转移到离心管中短时低速离心，然后用PBS离心洗涤两遍，并用500μLPBS重悬，使用流式细胞分析仪进行检测，测试前要将细胞吹打均匀。对于HepG2细胞：空白细胞的荧光吸收强度为1.32％，经包载钙黄绿素的mPEG-STlipos作用后的细胞荧光吸收强度为1.88％，经包载钙黄绿素的AGA-PEG2000-STlipos作用后的细胞荧光吸收强度为20％。对于A549细胞：空白细胞的荧光吸收强度为11.5％，经包载钙黄绿素的mPEG-STlipos作用后的细胞荧光吸收强度为28.9％，经包载钙黄绿素的AGA-PEG2000-STlipos作用后的细胞荧光吸收强度为35.0％。In order to further study the endocytosis of cells to liposomes loaded with calcein, the liposomes (referred to as mPEG-STlipos) that do not contain acetylglycyrrhetinic acid-polyethylene glycol-stearic acid and the liposomes containing acetylglycyrrhetinic acid The liposomes of glycyrrhetinic acid-polyethylene glycol-stearic acid (referred to as AGA-PEG2000 -STlipos) were respectively loaded with the model drug-calcein, and they were incubated with HepG2 cells and A549 cells for 8h (blank cells as a control), the final concentration of calcein was 2 μg/mL. Then aspirate the culture solution, wash the culture plate three times with PBS (0.01M, pH=7.4) buffer solution, add trypsin to digest to detach the cells, stop the digestion with serum-free medium, and transfer the digestion solution to a centrifuge tube Centrifuge at a low speed for a short time, then wash twice with PBS, resuspend with 500 μL PBS, and use a flow cytometer for detection. The cells should be blown evenly before the test. For HepG2 cells: the fluorescence absorption intensity of blank cells is 1.32%, the fluorescence absorption intensity of cells treated with mPEG-STlipos loaded with calcein is 1.88%, and the cells treated with AGA-PEG2000 -STlipos loaded with calcein The fluorescence absorption intensity was 20%. For A549 cells: the fluorescence absorption intensity of blank cells is 11.5%, the fluorescence absorption intensity of cells treated with mPEG-STlipos loaded with calcein is 28.9%, and the cells treated with AGA-PEG2000 -STlipos loaded with calcein The fluorescence absorption intensity was 35.0%.
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