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多西紫杉醇/壳聚糖靶向载药系统的制备及抗肿瘤活性研究
张恩惠
学位类型博士
导师李鹏程
2017-05-09
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词壳聚糖 羧甲基壳聚糖 多西紫杉醇 纳米粒 抗肿瘤
摘要胃癌是一种常见的消化道恶性肿瘤,我国每年有40万的胃癌新发病例,占全球40%左右,在我国是死亡率高达第二位的癌症。目前胃癌常见的治疗手段为化疗、放疗和手术治疗。传统胃癌化疗药物多西紫杉醇在临床应用中存在很多问题,其水溶性差、靶向性不足的缺点易引起患者出现多种严重的不良反应。壳聚糖是从虾蟹壳中提取的生物高分子聚合物,也是自然界中唯一的碱性多糖,有着良好的生物相容性,生物可降解性,无毒以及低致免疫性,这些特点使其成为一种有潜力的药物载体。随着纳米技术的发展,制备以壳聚糖为载体的多西紫杉醇纳米制剂可增加其水溶性和靶向性,提高生物利用度并增强抗肿瘤效果。
本文共分为两部分,首先以GX1作为胃癌靶向分子,N-去氧胆酸羟乙基壳聚糖作为纳米药物载体,多西紫杉醇作为抗肿瘤血管新生药物制备一种胃癌靶向纳米粒,并对纳米粒进行了结构表征,初步分析了药物的缓释机理,对纳米粒的抗肿瘤效果进行了体外和体内评价;其次,采用O-羧甲基化,N -羧甲基化,N,O-羧甲基化三种不同羧化位点的壳聚糖,以多西紫杉醇为模型药物,制备三种羧甲基壳聚糖载药纳米粒,对纳米粒进行结构表征及体外药理作用的比较,考察不同羧化位点对纳米药物的影响。得出以下主要结论:
(1)所制备的胃癌靶向纳米粒(GDD)包封率和载药量可达52.7%和24.6%,平均粒径为150.9 nm,zeta电位为16.0 mV,GDD在SEM和TEM下呈现类球形结构,表面光滑,分散均匀,无明显的聚集现象。GDD中多西紫杉醇的体外释放为pH响应性缓释释放,即在酸性pH下释放速度较中性pH下快,药物扩散类型为non-Fickian扩散。体外药理作用研究显示GX1可以提高GDD对Co-HUVEC细胞的摄取率,并且GDD在50 μM(多西紫杉醇浓度)以下的浓度对Co-HUVEC细胞的毒性显著强于原药多西紫杉醇。体内研究显示GDD能显著抑制裸鼠移植胃肿瘤的生长速度,抗肿瘤效果显著强于原药多西紫杉醇,且荷瘤裸鼠对GDD具有良好的耐受性,肿瘤切片TUNEL和HE染色结果显示GDD可以诱导大量肿瘤细胞发生凋亡。
(2)采用离子交联制备了O-羧甲基化,N-羧甲基化,N,O-羧甲基化三种不同羧化位点的壳聚糖载药纳米粒。通过考察羧甲基壳聚糖和交联剂氯化钙的用料比,优化了三种纳米粒的制备工艺。其中O-羧甲基壳聚糖纳米粒的包封率和载药量最大,N,O-羧甲基壳聚糖纳米粒的zeta电位的绝对值最大,三种纳米粒的平均粒径均可达到200 nm以下,且粒径分布均匀。体外药物释放实验显示O-羧甲基壳聚糖纳米粒的释放速度最快,释放量最大。药理作用研究显示O-羧甲基壳聚糖纳米粒对SGC7901细胞的毒性最强,而N-羧甲基壳聚糖纳米粒和N,O-羧甲基壳聚糖纳米粒的细胞毒性无显著性差异,当多西紫杉醇的浓度在50 μM以下时,三种纳米粒的细胞毒性均强于原药多西紫杉醇。荧光显微镜下观察发现三种纳米粒均能够被SGC7901细胞大量摄取,采用流式细胞仪测定细胞摄取率,发现O-羧甲基壳聚糖纳米粒的细胞摄取率最大,细胞摄取率的高低可能与壳聚糖2位未取代氨基的数量有关。
本文制备的新型GX1介导的多西紫杉醇/壳聚糖纳米粒,其抗肿瘤作用显著强于多西紫杉醇原药,为克服多西紫杉醇药物缺点,提高药物抗肿瘤效果的研发提供了新思路。本文还优化了离子交联法制备羧甲基壳聚糖纳米粒的条件,分析了不同羧化位点对羧甲基壳聚糖纳米粒的物化性质及药理作用的影响,发现O-羧甲基壳聚糖纳米粒的抗肿瘤活性和细胞摄取率最优,为壳聚糖抗肿瘤药物的开发提供理论依据。
其他摘要Gastric cancer (GC) is a common cancer in digestive system. In China, there are approximately 400 thousand cases annually which accounts for 40% of cases of the world and the death rate of GC is in the second place. The common treatment methods for GC are chemotherapy, radiotherapy and surgery. There are many problems in the clinical use of docetaxel, a traditional chemotherapeutic agent for GC. Docetaxel has very poor water-solubility and low targeting ability, which can cause many severe side effects in patients. Chitosan is a kind of macromolecular polymer extracted from shrimp and crab shells and the only alkaline polysaccharide in nature. Chitosan has good biocompatibility, biogradation, non-toxicity and low immunogenicity, which makes it a potential drug carrier. As the nanotechnology develops rapidly, the nanoformulation of docetaxel/chitosan could increase its water solubilty and targeting ability, enhance the bioavailability and anti-tumor effects.
This thesis is divided into two parts. Firstly, a new GC-targeted nanoparticle was prepared. Sructurally, GX1 was the GC vascular-targeted molecule and N-deoxycholic acid glycol chitosan was the nano-carrier encapsulating docetaxel, a anti-angiogenesis agent. Different characteristics of nanoparticles were determined and the drug release mechanism was analyzed. Subsequently, the in vitro and in vivo anti-tumor activities were determined. Secondly, three carboxymethyl chitosans with different carboxymethyl sites were synthesized as nano-carriers for docetaxel. The characteristics and in vitro anti-tumor activity of three kinds of nanoparticles were determined. The influence of carboxymethyl sites on nanoparticles was analyzed. The conclusion is as bellows:
(1) The GC-targeted nanoparticle (GDD) had encapsulating efficiency of 52.7% and drig loading content of 24.6%. The average size was 150.9 nm and zeta potential was 16.0 mV. GDD was spherical-shaped under TEM and SEM and the surface was smooth and distributed uniformly without accumulation. The release profile of docetaxel from GDD was pH-sensitive controlled release. The release rate under acid pH was significantly faster than that under neutral pH and the release mechanism analysis showed it was non-Fickan diffusion. The in vitro anti-tumor activity study showed that the presence of GX1 could enhance the cellular uptake of GDD in Co-HUVEC cells and GDD had significantly stronger cytotoxicity against Co-HUVEC cells than docetaxel free drug under the docetaxel concentration of 50 μM. The in vivo study showed that GDD could inhibited the growth of xenografted human gastic tumor in nude mice. The mice had high tolerance for GDD. The TUNEL and HE staining of tumor biopsies showed that GDD induced apoptosis of large amount of tumor cells.
 (2) Three docetaxel-loaded chitosan with different carboxymethyl sites based (O-carboxymethyl, N-carboxymethyl and N,O-carboxymethyl) nanoparticles were prepared by ionic-crosslinking. The preparing condition of each kind of nanoparticles was optimized by investigated the proportion of carboxymethyl chitosan and CaCl2, the crosslinking agent. The O-carboxymethyl chitosan nanoparticles had the highest encapsulating efficiency and drug loading content and N,O-carboxymethyl chitosan nanoparticles had the largest abosolute zeta potential value. The average sizes of three different nanoparticles were under 200 nm and the size distribution was uniform. The drug release rate and amount of O-carboxymethyl chitosan nanoparticles were the largest among them. The in vitro cytotoxicity showed that the anti-tumor activity against SGC7901 cells of O-carboxymethyl chitosan nanoparticles was strongest, while there was no significant difference between O-carboxymethyl chitosan nanoparticles and N,O-carboxymethyl chitosan nanoparticles. When the concentration of docetaxel was under 50 μM, the cytotoxicity of three nanoparticles was stronger than the free drug. Furthermore, high cellular uptake of three nanoparticles in SGC 7901 cells was observed under fluorescent microscope. The celluar uptake of O-carboxymethyl chitosan nanoparticles was the most as measured by the flow cytometry, which maybe related to the amount of unsubstituted amine groups.
In this thesis, the preparation and characterization of a new GX1-targeting docetaxel/chitosan nanoparticle were described and the in vitro and in vivo anti-tumor activities were determined. GDD exhibited stronger activity than the free drug docetaxel, which provided a new idea for the development of docetaxel nanoformulations with potential clinical use in the future. Besides, after optimizing the preparation condition of carboxymethyl chitosan nanoparticles by ionic-crosslinking, the influence of carboxymethyl sites on physical-chemical characteristics and anti-tumor activity of carboxymethyl chitosan nanoparticles was analyzed. Results showed that O-carboxymethyl chitosan nanoparticles had the strongest anti-tumor activity and the largest cellular uptake, which provided evidence for the development of chitosan-based anti-tumor drugs.
学科领域海洋科学 ; 海洋生物学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136656
专题实验海洋生物学重点实验室
作者单位1.中国科学院大学
2.中国科学院海洋研究所
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张恩惠. 多西紫杉醇/壳聚糖靶向载药系统的制备及抗肿瘤活性研究[D]. 北京. 中国科学院大学,2017.
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