【作者】 郭波红；

【导师】 程怡；

【作者基本信息】 广州中医药大学 ， 中药学， 2010， 博士

【摘要】 脂质体是一种由磷脂双分子层构成的具有水相内核的封闭囊泡。脂质体具有良好的生理相容性,可赋予药物良好的靶向性能,改变药物分布规律,提高药物的疗效和治疗指数,是抗肿瘤药物的理想传输载体。通过表面修饰可以改变脂质体的性质和生物学行为：在脂质体表面修饰特异性的配基（如抗体、多肽、叶酸、糖基）,能使脂质体具有主动靶向的特性。受体介导系统（receptor-mediated endocytosis systems, RME systems）是指针对肝细胞膜上的不同受体,对药物或载体进行修饰,利用配体与肝细胞受体的高特异性,高选择性和高亲和性,将药物导向到肝细胞内发挥药理作用的给药系统。去唾液酸糖蛋白受体（asialoglycoprotein receptor, ASGPR）是肝细胞膜上表达丰富的一种内吞性受体,存在于肝脏实质细胞朝向窦状隙一侧的细胞膜上。哺乳动物肝细胞表面特异存在的ASGPR,能专一识别末端带有半乳糖残基或N-乙酰半乳糖胺基的糖链,并能与之特异性结合,通过肝细胞内吞作用,可将其配体运送至溶酶体内进行降解。利用ASGPR能选择性吞噬异源性复合物的特性,以该受体为靶点,可将药物特异性导入肝细胞治疗乙型肝炎和高血脂等疾病,或直接导入肝癌细胞治疗原发性肝癌。甘草次酸（glycyrrhetinic acid, GA）是甘草酸的苷元,口服后经水解或β-葡萄糖醛酸酶分解而成。甘草次酸的溶解度极小,口服几乎不吸收,普通注射剂的生物利用度低。已知甘草次酸具有明显的抗炎、抗病毒、抗肿瘤和防癌作用,临床上常用来治疗慢性肝炎及肝癌等。此外,研究还发现GA有提高内耳听力抗缺氧、抗乙酰胆碱脂酶、抗心律失常的作用,并具有抗HIV活性。同时在肝脏病的应用研究中也取得了突破,GA的保肝及抑制肝癌的基本机理是通过对肝细胞的吸附能力对抗肝毒性化合物。但是,该类药物长期服用可引起类醛固酮增多症,其特征是钠潴留、钾排泄增多,从而导致水肿、高血压、四肢瘫痪和低血钾症等。为了降低上述毒副作用以及改善甘草次酸的溶解性、吸收性等,本实验将其制备成脂质体,可以增加其水溶性,并使其具有一定的靶向性。本课题选择中药抗癌有效成分甘草次酸为模型药物,以半乳糖作为靶向头基,制备了ASGPR介导的主动靶向脂质体。通过配体与肝脏表面去唾液酸糖蛋白受体的相互作用,可介导脂质体靶向至肝癌细胞,实现对肝癌细胞的主动靶向。首先用酶促合成法合成了脂质体肝靶向载体N-十八烷基-4-[（D-吡喃半乳糖基）氧基]-2,3,5,6-四羟基己酰胺,目前该酰化主要采用化学法,存在区域选择性差、反应步骤复杂、环境不友好等诸多问题。本论文探讨了酶促酰胺化反应的可行性,并对比研究了不同反应介质、不同脂肪酶催化反应的影响规律,建立了可用于高效、高区域选择性地合成N-十八烷基-4-[（D-吡喃半乳糖基）氧基]-2,3,5,6-四羟基己酰胺的生物催化反应体系。通过TLC、IR、HPLC-MS、1H-NMR、13C-NMR、DSC等初步鉴定为目标产物,符合试验要求。对甘草次酸进行了处方前研究,包括建立体外甘草次酸HPLC含量测定方法、油水分配系数和包封率测定方法。GA理化性质研究表明,GA在水中难溶,溶解度仅约6.32μg/mLo其在正辛醇中易溶,脂溶性强。通过对脂质体制备方法筛选,以包封率和性状特征为指标,最终确定采用乙醇注入法制备脂质体,并通过单因素优选和正交试验,优选最佳处方。结果表明,甘草次酸的lgP值在4.69左右,是一种脂溶性极强的化合物,适合制备脂质体,采用葡聚糖凝胶法测定脂质体的包封率,游离药物与含药脂质体分离效果好,柱回收率满足要求,且简单快速。最终确定GA肝靶向脂质体的处方如下：固定N-十八烷基-4-[（D-吡喃半乳糖基）氧基]-2,3,5,6-四羟基己酰胺的比例为混合类脂的10%（摩尔比）,磷脂、胆固醇、十八胺、GA以摩尔比7：5：1：2,分散介质为pH 7.4磷酸盐缓冲液（PBS） （33.23 mmol/L）,制成10 mL。乙醇注入法避免使用有毒的有机溶剂,产品性能稳定,包封率>80%,粒径大小适中并符合正态分布。对甘草次酸肝靶向脂质体的理化性质进行系统考察,主要包括粒径、形态学、Zeta电位、体外释放度、包封率测定、稳定性考察及乙醇残留量测定等。结果表明,甘草次酸肝靶向脂质体外观呈半透明略带乳光的混悬液,色泽均匀,流动性好。通过透射电镜观察,脂质体微粒大小均匀,圆整,分散性好。脂质体样品的粒径分布范围较窄,平均粒径195 nm,呈单峰分布；用凝胶柱分离法测定包封率,测定结果在80%以上；氧化指数低,化学稳定性考察结果表明氧化程度小；放样稳定性结果表明,NOH-GA-LP在（0-4）℃条件下放置3个月仍能保持稳定；稀释稳定性表明,将其按临床用法稀释,可以保持稳定,没有药物的渗漏和沉淀。体外释放试验结果表明,具有明显的缓释作用,体外释放曲线符合Higuchi方程,乙醇残留量测定结果小于0.5%。进一步观察甘草次酸肝靶向脂质体的肝靶向特性,分别采用SD大鼠进行药物动力学试验和昆明种小鼠进行组织分布试验。在建立血浆和组织中药物测定方法的基础上,按一定的时间间隔目眦静脉采血,处理样品进行HPLC分析。药动学数据如下：GA溶液、GA-LP和NOH-GA-LP的t1/2分别为3.91、46.49和50.18 h；AUC（o₂4）分别为1.95、12.85和15.41μg·h/mL:MRT（o-24）分别为1.75、8.18和8.11 h。GA-LP及NOH-GA-LP的平均滞留时间（MRT）分别为GA溶液的4.7及4.6倍,表明脂质体较药物溶液有明显的缓释效果。在动物体内分布研究中,以甘草次酸溶液为对照测定了脂质体经小鼠尾静脉注射后,不同时间点药物的体内分布情况。GA-LP及NOH-GA-LP对肝脏的相对靶向效率分别为甘草次酸对照溶液的2.4及4.83倍,表明半乳糖配体的引入提高了脂质体对肝脏的靶向性。体外药效学评价采用MTT法观察不同比例配体的脂质体对HepG2细胞的抑制作用,结果显示,细胞生长显著受抑,50μmol/L时抑制率达96%,抑制率与GA浓度呈正相关,不同比例配体的脂质体对HepG2细胞的抑制作用有显著性差异（P<0.01）,含10%配体的脂质体对HepG2细胞的抑制作用最强,表明半乳糖化脂质体可以有效抑制肝癌细胞HepG2细胞生长。综上所述,本课题采用了酶促法进行半乳糖的修饰,生成了一个能够接载在脂质体表面的肝靶向载体。以包封率作为指标,用乙醇注入法制备了符合药典要求的、具有主动靶向作用的甘草次酸肝靶向脂质体,制备工艺稳定,粒径、包封率和渗漏率等指标皆符合要求。建立了定量测定药物浓度的HPLC法以及体内生物样品测定的HPLC法。对比研究了GA-LP与NOH-GA-LP体外释药规律,进而研究了大鼠体内药动学和小鼠体内分布规律,并对HepG2细胞的抑制作用进行了研究。通过这些研究,探讨了酶促酰化反应生成肝靶向载体的可行性,为探讨酶促修饰脂质体给药系统奠定了理论与实践基础。同时,该研究丰富了脂质体纳米粒给药系统的研究内容,该研究不仅具有较高的学术参考和借鉴,而且实验结果证实该设想具有一定的可行性,为癌症的治疗提供新思路和新手段。更多还原

【Abstract】 Liposomes are composed of phospholipid bilayer structures that encapsulate an aqueous interior. Liposomes are promising and effective vector in tumor treatment. Liposomal antineoplastic agents are characterized by targeting tumor and controlled-release, and thus they can increase therapeutic index, improve clinical effect and reduce toxicity. Liposome properties and biological behaviors can be controlled by surface modification. Attachments of specific ligands, such as monoclonal antibody, peptides, folate and galactosyl, make liposomes own active targeting effeet to specific cells or tissues.It was reported that asialoglycoprotein receptors (ASGPR) were expressed plentifully on the surface of hepatoma cells, and targeting could be accomplished through introduction of galactose residues which can bind specifically to the ASGPR on hepatoma cells, into drug carriers for the treatment of liver cancers. There are large amount of ASGPR in the mammalian hepatocytes, deasialylated proteins can be bound and internalized in the cell interior. Terminal P-D-galactose or N-acetylgalactosamine residues can be recognized by ASGPR and they were considered as specially targeting ligands for drug carriers.Glycyrrhetinic acid is an active principal aglycone of glycyrrhizin which has been shown to be hydrolyzed by glucuronidase in intestinal bacteria after oral administration. Glycyrrhetinic acid is a lipophilic drug with a very low solubility in water (<0.01 mg/ml), which may result in its poor bioavailability. After absorption in gut, glycyrrhetinic acid is eliminated fast in plasma. Glycyrrhetinic acid has been shown to possess several beneficial pharmacological activities, such as anti-inflammatory activity, direct and indirect antiviral activity, and an antihepatitis effect. Glycyrrhetinic acid may cause sodium retention and potassium loss, which are associated with hypertension while the adverse effects of glycyrrhetinic acid seem to be dose-dependent.In this research, glycyrrhetinic acid was chosen as the model drug and galactose as the targeting head. Asialoglycoprotein receptor-mediated active targeting liposomes were prepared. Liposomes could be targeted to liver through the interaction between ligand and receptor on the carcinoma cells of liver. The drug delivery system actively targeting to malignant liver cells is expected to be achieved.Lipid materials for preparing liver targeting liposomes, such as N-octadecyl-4-[(D-galactopyranosyl)oxy]-2,3,5,6-tetrahydroxy hexanamide (NOH), were enzymic synthesized. Most of the present strategies for the amidation were focused on chemical methods, which were hampered by the low regioselectivity, the tedious reaction procedures and the environmental concerns of the process. The possibility of the enzymatic regioselective acylation was explored in this dissertation. Also, the effects of some variables on the enzymatic reaction in different media and lipases were characterized. Additionally, novel enzymatic reaction systems which could be used for highly efficient and regioselective preparation of monoesters of N-octadecyl -4-[(D-galactopyranosyl)oxy]-2,3,5,6-tetrahydroxy hexanamide have been well established. The synthetic products were determined via TLC, IR, HPLC-MS,’H-NMR,13C-NMR, DSC etc.The pre-prescription of glycyrrhetinic acid was carried out via three steps: establishment of glycyrrhetinic acid by HPLC analysis method, oil-water distribution coefficient and determination method of encapsulation efficiency. The study of physico-chemical property showed that glycyrrhetinic acid was a poorly water-soluble drug. Its solubility in water was only about 6.32μg/mL. It was easy to dissolve in n-octanol and had a good liposolubility. After choosing several common methods of preparation of liposomes, such as film dispersion method and ethanol injection method, encapsulation efficiency and trait characteristics as indicators, liposomes were prepared by ethanol injection technique. The lgP value of glycyrrhetinic acid was about 4.69. Gglycyrrhetinic acid was high lipophilic compounds, which adapt to preparation of liposomes. The unencapsulated glycyrrhetinic acid and liposomes were separated by sephadex gel G-50, the encapsulation efficiency was detected by HPLC. Through a single factor and orthogonal design optimization, we achieve the best formulation. Ethanol injection technique is a good process to avoid using of toxic organic solvents, and the products have shown characteristic and good stability, fit encapsulation efficiency, appropriate particle size and be in accordance with the normal distribution.To observe the glycyrrhetinic acid liver targeting liposomes physical and chemical properties systematically, including particle size, morphology, Zeta potential, in vitro release rate, determination of encapsulation efficiency, stability reseach, determination of alcohol residue, and so on. The appearance of glycyrrhetinic acid liver targeting liposomes was slightly translucent opalescence, color uniformity, good mobility. The morphological examination of glycyrrhetinic acid liver targeting liposomes was performed using transmission electron microscopy. The liposomes were spherical or ellipsoidal shape. The particle size and Zeta potential of the liposomes were measured. the mean partical size of 195 nm, and Zeta potential of 38.7 mv. The unencapsulated glycyrrhetinic acid and liposomes were separated by sephadex gel G-50, the encapsulation efficiency was detected by HPLC. Entrapment efficiency is over 80%. Resarch on chemical stability show that oxidation index is low. Reseach on lofting stability results show that glycyrrhetinic acid liver targeting liposomes situated at (0-4)℃place for three months to maintain stability. The stability of glycyrrhetinic acid liver targeting liposomes was better. The release kinetics in vitro obeyed Higuchi equation. the glycyrrhetinic acid liver targeting liposomes have obviously sustained effect. Alcohol residue is under 0.5%.Furthermore, the pharmacokinetic behavior and distribution of glycyrrhetinic acid liver targeting liposomes were studied. Both the two formulations and free glycyrrhetinic acid were administered via tail vein. Glycyrrhetinic acid was separated from the plasma component by solvent extract. The plasma concentration and tissue distribution of the drug was determined by HPLC. Pharmacokinetic parameters of rats were as follow:t1/2 of GA, GA-LP and NOH-GA-LP were 3.91,46.49 and 50.18 h, respectively; AUC(0-24) of them were 1.95,12.85 and 15.41μg-h/mL, respectively; MRT(0-24) of them were 1.75,8.18 and 8.11 h, respectively. HPLC was employed to exmaine the tissue distribution of mice at diffetent times after liposomes were injected into their tail veins. Compared with glycyrrhetinic acid control solution, the relative targeting efficiencies of liver of glycyrrhetinic acid, GA-LP and NOH-GA-LP were 2.4 and 4.83 respectively, which indicated that the efficiency of liver targeting was improved when galactose ligand was introduced to liposomes.HepG2 cells were cultured in vitro, and the tumor cell inhibition test was conducted with MTT assay. The result showed that 10 %NOH-GA-LP was significantly more suppressive than the 5%NOH-GA-LP. There was significantly different between the 5%NOH-GA-LP and 10%NOH-GA-LP with MTT assay. HepG2 cells were suppressed when galactose ligand was introduced to liposomes.In conclusion, glycyrrhetinic acid active targeting liposomes were successfully prepared by using ethanol injection method and the method is repeatable with satisfactory results. Physical and chemical stability, particle size, encapsulation efficiency and the leakage rate of the products meet the requirements and so on. Glycyrrhetinic acid in NOH-GA-LP and in biological material was detemrination by HPLC. A good theoretical and practical foundation was established for the exploration of the liposomes’ dosage formula, characteristics and work mechanism in this research, which not only enlarged research field of liposomes but also brought new ideas and new methods to treat cancer. Further more, this research has potential value in clinic practise.更多还原