【作者】 张正；

【导师】 裴瑾；

【作者基本信息】 吉林大学 ， 生物药学， 2016， 硕士

【摘要】 肺癌是最常见的肺原发性恶性肿瘤,也是发病率和死亡率增长最快,对人群健康和生命威胁最大的恶性肿瘤之一。绝大多数肺癌起源于支气管黏膜的上皮细胞。目前临床上吉西他滨是治疗晚期非小细胞肺癌的一线药物,但因其半衰期短需大剂量持续给药维持体内有效的药用浓度。这种大剂量的给药方式给患者带来了很强的毒副作用,限制了其临床效用。本课题旨在,利用肺癌细胞表面过表达的雌激素受体(Estrogen receptors,ER)与配体雌酮(Estrogen,ES)间的特异性高亲和力,研制出一种新型以雌酮修饰的长效吉西他滨脂质体制剂,以提高药物对肿瘤的靶向性同时能够实现在体内达到长效循环的效果。此外,本论文对该新型脂质体制剂进行了表征、体外靶向性、体外细胞毒及体内抗肿瘤的研究。首先采用硫酸铵梯度法制备4种不同的吉西他滨脂质体制剂,即吉西他滨普通脂质体(L-GEM)、吉西他滨靶向脂质体(ES-L-GEM)、吉西他滨长效脂质体(SSL-GEM)和吉西他滨长效靶向脂质体(ES-SSL-GEM)。然后分别对这四种脂质体制剂进行表征研究,通过透射电镜对脂质体形态进行观察,采用动态光散射粒径分析仪测定粒径大小、粒径分布、多分散系数和Zeta电位,借助紫外可见分光光度计检测吉西他滨脂质体的包封率,并通过透析法检测四种制剂储存在4℃和25℃条件下于不同时间段的稳定性。之后在荧光显微镜下观察肺癌细胞A549对加载了罗丹明荧光染料(Rh B)的四种脂质体制剂的摄取情况,以此验证长效靶向脂质体的体外靶向作用。为探究A549细胞对长效靶向脂质体的摄取机制,于吉西他滨脂质体与细胞孵育前加入内吞抑制剂:蔗糖、染料木黄酮、盐酸阿米洛利等,并于荧光显微镜下观察内吞结果。最后采用MTT比色法对吉西他滨及所制备的的4种脂质体制剂分别进行细胞毒实验,以此观察体外抗肿瘤作用。实验结果显,成功制备出四种脂质体制剂(L-GEM、ES-L-GEM、SSL-GEM、ES-SSL-GEM),粒径约在120nm~130nm间,粒径较为均一,PDI在0.1~0.2范围内,脂质体并带有一定负电荷。吉西他滨长效靶向脂质体的稳定性显著优于吉西他滨靶向脂质体和普通脂质体,并且在4°C下保存至48h后吉西他滨长效靶向脂质体制剂的渗漏率不超过10%。体外靶向性的研究结果证实ES-L-Rh B与ES-SSL-Rh B均对A549细胞具有主动靶向作用,且ES-SSL-Rh B具有长效作用。此外,除受体介导的内吞作用外,巨胞饮介导的内吞作用是长效靶向脂质体进入细胞的主要途径。吉西他滨及所制备的这四种脂质体对A549细胞的毒性作用由强到弱依次为:ES-SSL-GEM、SSL-GEM、ES-L-GEM、L-GEM、GEM,且ES-SSL-GEM的抗肿瘤效果显著高于其他四种制剂,在体内实验中也可以看出ES-SSL-GEM抗肿瘤效果是最好的。本课题在常规脂质体中载入特异性识别雌激素受体的雌酮片段和长效PEG分子,成功研制出一种具有长循环和肿瘤特异性靶向特性的高效低毒的脂质体载药系统,将这种载药系统携带临床上的一线抗肺癌用药物吉西他滨,针对有高表达雌激素受体的非小细胞肺癌进行各种研究,发现这种吉西他滨长效靶向脂质体能够延长药物在体内的作用时间,药物对肿瘤细胞的杀伤作用随着浓度和时间的增加而提高,其靶向性也可显著增强其对肿瘤的定位作用并降低了毒副作用,有较高的临床价值。希望本论文所制备的吉西他滨长效靶向脂质体在不久的将来可以有效的应用于肺癌的临床治疗。更多还原

【Abstract】 Lung cancer is one of the most malignant tumors, which occurs mostly in bronchial epithelial cells. Gemcitabine(GEM) is a common anti-tumor drug used in the first-line chemotherapy for advanced non-small cell lung cancer. Due to its short half-life period Gemcitabine must be applied in high-dose that results accumulation of the toxicities.This study aims to design a new drug transfer system to increase the concentration of Gemcitabine in tumor cells and decrease the side effects for normal tissues. As artificial double phospholipids, liposome is able to extend the half-life period of drug in vivo. Together with estrogen receptors(ER), which are widely overexpressed on non-small cell lung cancer cells and act as a potential target for cancer targeted therapy, the modified drug loaded liposomes could specifically target tumor cells and further release chemotherapeutic drugs.Four types of Gemcitabine loaded liposomes(GEM loaded conventional liposomes L-GEM, GEM loaded targeted liposomes ES-L-GEM, GEM loaded sterically stabilized liposomes SSL-GEM and GEM loaded sterically stabilized and targeted liposomes ES-SSL-GEM) were prepared via thin film hydration method.The physicochemical characteristics of these four GEM loaded liposomes were investigated, including the morphological examination, particle size, zeta potential,encapsulation efficiency, drug-loading capacity and the stability at 4°C and 25°C. To evaluate the in vivo target effect, the fluorescence intensity of lung cancer cells A579 treated with Rhodamine B(Rh B) loaded liposomes was measured by fluorescence microscope. By adding different inhibitory agents of endocytosis including sucrose,genistein, amiloride hydrochlorid and ES, the mechanism of cellular uptake wasdetermined. Furthermore, the in vitro anti-tumor efficacy of GEM and the prepared GEM-loaded liposomes was investigated via MTT assay.We observed that, the successfully prepared four types of GEM loaded liposomes obtained average particle sizes in the range of 120nm~140nm and PDI in the range of 0.1~0.2. The zeta potentials of the liposomes showed negative. After storing at 4°C for 48 h SSL-GEM and especially ES-SSL-GEM were obviously more stabile than other liposomes. The in vitro targeting test revealed that,ES-L-Rh B and ES-SSL-Rh B were able to active target A549 cells, and that ES-SSL-Rh B showed sustained release effect. The result of inhibition assay of cellular uptake confirmed that, the cellular uptake of liposomes mainly depended on caveolin endocytosis and estrogen receptor-mediated endocytosis. Compare to Gemcitabine and other GEM-modified liposomes, ES-SSL-GEM, which benefited from its sterical stability and ER targeting speciality, obtained the greatest in vitro anti-tumor efficacy, In vivo experiments can be seen that the ES-SSL-GEM ’s anti-tumor effect is the best one.In this study, we designed and successfully prepared a new type of Gemcitabine loaded sterically stabilized and ES-modified liposome ES-SSL-GEM, which was able to specifically target the non-small cell lung cancer cells and showed high anti-tumor efficiency. We expect that, in the near future this new Gemcitabine loaded liposome could be applied to clinical treatment and support the lung cancer therapy.更多还原