【作者】 金成；

【导师】 郭国祯；

【作者基本信息】 第四军医大学 ， 军事预防医学， 2007， 博士

【摘要】 肿瘤乏氧细胞的放射抗拒性问题一直是放射肿瘤学和放射生物学研究的重点之一。乏氧细胞放射增敏剂被认为是最有希望解决这个难题的方法,各国医学工作者对此进行了深入的研究,已研制出了多种类型的增敏剂,如亲电子硝基化合物、生物还原活性物、巯基抑制剂和修复抑制剂等。但是,这些增敏剂的神经毒性、消化道症状等不良反应限制了它们的临床应用。目前认为联合用药是比较现实、稳妥、易行的提高增敏效果并减低毒性的途径,国外已有较多相关报道。本课题选用依他硝唑和紫杉醇两种有代表性的放射增敏剂,研究其联合应用的协同作用,为临床放射治疗增敏提供一种新的给药组合。同时,为了进一步克服放射增敏剂的不良反应并提高疗效,我们对它们的剂型进行改进,引入纳米技术。利用纳米药物载体对药物的可控制释放、靶向传输、提高难溶药物的溶解率和吸收率、提高药物疗效并降低毒副作用等优点,来充分发挥依他硝唑和紫杉醇的增敏效果,为放射增敏剂的临床应用提供新的思路。目的:研究依他硝唑和紫杉醇两种放射增敏剂的协同作用,制备出具有生物活性并可控释药物的依他硝唑/紫杉醇纳米粒,明确载药纳米粒增敏效果和剂型优势。方法:1.乏氧肿瘤细胞(人乳腺癌MCF-7细胞和人宫颈癌HeLa细胞)经依他硝唑和/或紫杉醇处理后接受60Coγ射线照射,采用MTT检测增殖能力,流式细胞仪检测细胞周期,平板克隆形成实验检测细胞克隆形成能力,比较单独用药与联合用药的差异;2.采用单乳和复乳溶酶挥发法制备单独和联合包裹依他硝唑/紫杉醇的纳米粒,高效液相色谱分析纳米粒的载药率、包封率和模拟体外释药,激光衍射仪研究粒径大小及分散范围,扫描电镜分析纳米粒的形态;3.乏氧MCF-7和HeLa细胞分别接受药物单体和载药纳米粒(根据体外释药曲线计算出释药量)作用一定时间后,观察给药后细胞形态的变化,透射电镜和荧光显微镜观察肿瘤细胞对纳米粒的吞噬摄取。此后肿瘤细胞接受60Coγ射线照射,采用流式细胞仪检测细胞周期,平板克隆形成实验检测细胞克隆形成能力,研究载药纳米粒的增敏活性并比较增敏效果的差异。结果:1.依他硝唑和紫杉醇联合应用后,照后5 d内MTT检测未观察到协同作用。依他硝唑和/或紫杉醇给药的乏氧HeLa细胞照后被阻滞在细胞周期的G1期,两种药物联合应用较单独用药导致更多的HeLa细胞停留在G1期,但对于乏氧MCF-7细胞周期无显著影响。细胞存活分析结果显示依他硝唑和/或紫杉醇可以增敏乏氧HeLa细胞,在吸收剂量为6、8和10Gy时两药具有协同作用;依他硝唑和/或紫杉醇也可以增敏乏氧MCF-7细胞,但两药无协同作用。100 nM紫杉醇对于乏氧MCF-7和HeLa细胞的辐射增敏作用较1 mM依他硝唑显著。HeLa细胞较MCF-7细胞对紫杉醇敏感。2.紫杉醇纳米粒呈光滑球形,粒径分布在100～500 nm之间,平均约300 nm,载药率和包封率分别为4.50%和85.51%,模拟体外释药曲线呈双相,即在爆发释放之后为缓慢释放,14 d内药物释放约为30%, 1 d内释放量占15%。紫杉醇纳米粒作用后,MCF-7细胞形态发生改变,极性增加,呈梭形;部分HeLa细胞出现皱缩,并且两种肿瘤细胞G2期细胞比例升高。透射电镜和荧光显微镜观察到细胞对纳米粒的吞噬摄取。给药组乏氧MCF-7和HeLa细胞照射后克隆形成能力较对照降低,紫杉醇纳米粒组较紫杉醇单体组作用更明显。3.依他硝唑纳米粒呈光滑球形,粒径分布在90～190 nm之间,平均约120 nm,载药率和包封率分别为1.66%和18.02%,模拟体外释药曲线呈双相,爆发释放现象明显,3 h内释放量约占50%, 1 d内药物释放超过90%。透射电镜和荧光显微镜结果可见细胞对纳米粒的吞噬摄取。给药组乏氧MCF-7和HeLa细胞照射后的克隆形成能力较对照降低,依他硝唑纳米粒组较依他硝唑单体组作用更明显。4.紫杉醇纳米粒、依他硝唑纳米粒和紫杉醇+依他硝唑纳米粒被成功制备。三种纳米粒均呈光滑球形,粒径分布在80～150nm之间,平均约110 nm。紫杉醇纳米粒的载药率和包封率分别为4.53%和85.52%,依他硝唑纳米粒的载药率和包封率分别为1.86%和20.06%。在依他硝唑+紫杉醇纳米粒中,紫杉醇的载药率和包封率分别为4.62%和90.51%,依他硝唑的载药率和包封率分别为1.92%和23.16%,在紫杉醇+依他硝唑纳米粒中两种药物的包封率均较在单药载药纳米粒中略高。两种药的模拟体外释药曲线均呈双相,紫杉醇在5 d内药物释放约为25%,1 d内释放量占15%;依他硝唑在3 h内释放量约占50%,1 d内药物释放超过90%。在依他硝唑+紫杉醇纳米粒中,两种药物的体外释放量较在单药纳米粒中略高。荧光显微镜和透射电镜结果可见MCF-7和HeLa细胞吞噬摄取紫杉醇+依他硝唑纳米粒。三种载药纳米粒给药后,乏氧MCF-7和HeLa细胞照射后的克隆形成能力较对照均降低,其增敏作用强弱依次为:依他硝唑纳米粒<紫杉醇纳米粒<紫杉醇+依他硝唑纳米粒。结论:1.依他硝唑和紫杉醇联合应用对乏氧HeLa细胞具有协同放射增敏作用,本课题为临床放射治疗提供了一种新的给药组合。2.依他硝唑/紫杉醇纳米粒被成功制备,它们的表征满意,可以被肿瘤细胞吞噬摄取,有生物活性的药物从纳米粒中释放。在本实验条件下,载药纳米粒的增敏作用较药物单体明显,两种药物联合包裹的纳米粒较单独包裹的纳米粒作用显著。依他硝唑/紫杉醇纳米粒具有剂型优势,本课题为放射增敏剂的临床应用提供了新的思路。更多还原

【Abstract】 Radiation resistance of hypoxic tumor cells is one of the key points in radiation oncology and biology. It is thought that radiosensitizers are potential to overcome the problem. This area attracted the increasing attention from the medical researchers all over the world. Many kinds of radiosensitizers, such as electro-affinic compounds, biological reducers, SH inhibitors and reparation inhibitors etc., were developed. However, the side effects, such as neurotoxicity and symptoms of enteron etc., limited their clinical application. In order to increase radiosensitization without increasing toxicity, the combination of the drugs is actual, safe and effective. There were many correlative reports abroad. This study was carried out to determine the synergetic radiosensitizing effect of etanodazole and paclitaxel when administered together at clinically relevant concentrations. The study would provide a new combination of radiosensitizers to radiotherapy. Meanwhile, in order to increase further radiosensitization without increasing toxicity, the nanoparticles containing etanodazole and/or paclitaxel were prepared. The advantages of such a formulation include the controlled and targeted delivery of drugs, increased solubility and ingestion of drugs, increased therapeutic effect and reduced side effects. This study would provide a new idea for clinical application of radiosensitizers.OBJECTIVE:The object of this study was to determine the synergetic radiosensitizing effect of etanidazole and paclitaxel, to prepare active and controlled nanoparticles containing etanidazole and/or paclitaxel, to prove the radiosensitization of the drug-loaded nanoparticles and their advantage.METHODS:1. After administered together at clinically relevant concentrations of etanidazole and/or paclitaxel into the two hypoxic human tumor cell lines: a breast carcinoma (MCF-7) and a carcinoma cervicis (HeLa), cells were irradiated by 60Co gamma rays. The 3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay was used to determine the number of surviving cells. Cell cycle was evaluated by Flow cytometry (FCM). Cell viability was measured by the ability of single cell to form colonies in vitro. Difference between the combination of the drugs and drug alone was studied.2. The poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles containing etanidazole and/or paclitaxel were prepared by o/w and w/o/w emulsification-solvent evaporation method. The drug loading efficiency (LE), encapsulation efficiency (EE) and release profile in vitro were measured by high-performance liquid chromatography (HPLC). The size distribution and morphology of the nanoparticles were investigated by laser diffraction analyzer and scanning electron microscope (SEM).3. After administered the free drugs and the drug-loaded nanoparticles into hypoxic MCF-7 and HeLa cells (released drug doses calculated according to the release profile in vitro), the morphology of cells was photographed using phase-contrast photomicrographe. The cellular uptake of nanoparticles by MCF-7 and HeLa cells was evaluated by transmission electronic microscopy (TEM) and fluorescence microscopy. Then the tumor cells were irradiated by 60Co gamma rays. Cell cycle was evaluated by FCM. Cell viability was determined by the ability of single cell to form colonies in vitro. Radiosensitization of the drug-loaded nanoparticles and their difference were studied.RESULTS:1. The synergistic radiosensitive effect of etanidazole and paclitaxel was less obvious during 5 d after irradiation. After hypoxic HeLa cells administered were irradiated, the cells were mainly blocked in G1 phase. The combination of the two drugs resulted in more HeLa cells blocked in G1 phase. For MCF-7 cells, there was no significant statistical difference among the groups. The synergistic radiosensitizing effect of these two drugs on MCF-7 cells was not observed, but the radiosensitizing efficiency was additive for HeLa cells irradiated at 6, 8 and 10Gy. The radiosensitizing effect of paclitaxel at 100 nM was more significant than that of etanidazole at 1 mM. HeLa cells were more sensitive to paclitaxel than MCF-7 cells.2. The prepared paclitaxel-loaded nanoparticles were spherical shape with size between 100nm and 500nm, about 300nm on average. The loading efficiency of 4.50% and the encapsulation efficiency of 85.51% were obtained. The drug release pattern was biphasic with a fast release rate followed by a slow one. The amount of cumulated paclitaxel release over 14 days was about 30%. The kinetic data showed that majority of the drug release occurred in the first day (approximately 15%). Co-culture of MCF-7 and HeLa cells with paclitaxel-loaded nanoparticles demonstrated that MCF-7 cells extremely spreaded and became fusiform, and the part of HeLa cells treated was marcid. The number of cells blocked in the G2/M phase for two tumor cell lines significantly increased. The cellular uptake of nanoparticles was observed. The paclitaxel-loaded nanoparticles and free paclitaxel more effectively sensitized hypoxic tumor cells to radiation than control. The radiosensitization of paclitaxel-loaded nanoparticles was more significant than that of free paclitaxel.3. The prepared etanidazole-loaded nanoparticles were spherical shape with size between 90nm and 190nm, about 120nm on average. The loading efficiency of 1.66% and the encapsulation efficiency of 18.02% were obtained. The drug release pattern was biphasic with a fast release rate followed by a slow one. The burst effect was very large(about 50% in 3 h). The kinetic data showed that majority of the drug release occurred in the first day (approximately 90%). The cellular uptake of nanoparticles was observed. The etanidazole-loaded nanoparticles and free etanidazole more effectively sensitized hypoxic tumor cells to radiation than control. The radiosensitization of etanidazole-loaded nanoparticles was more significant than that of free etanidazole.4. The paclitaxel, etanidazole and paclitaxel+etanidazole nanoparticles were prepared. The surface of all the nanoparticles was smooth, rounded morphology and polydispersed with a diameter between 80 nm and 150 nm, about 110 nm on average. The results indicated that a loading efficiency of 4.53% and an encapsulation efficiency of 85.52% were obtained for paclitaxel-loaded nanoparticles, 1.86% and 20.06% for etanidazole-loaded nanoparticles. For paclitaxel+etanidazole nanoparticles, the loading efficiency and the encapsulation efficiency were 4.62% and 90.51% for paclitaxel, 1.92% and 23.16% for etanidazole, respectively. The drug encapsulation efficiency of paclitaxel+etanidazole nanoparticles is slightly higher than that of single drug-loaded nanoparticles. All the drug release patterns were biphasic with a fast release rate followed by a slow one. The amount of cumulated paclitaxel release over 5 days was about 25%. The kinetic data showed that majority of the drug release occurred in the first day (approximately 15%). The kinetic data showed that majority of the drug release from etanidazole-loaded nanoparticles occurred in the first day (approximately 90%). The burst effect was very large (about 50% in 3 h). The amount of cumulated drug release from paclitaxel+etanidazole nanoparticles was slightly more than that from single drug-loaded nanoparticles. The cellular uptake of paclitaxel+etanidazole nanoparticles was observed. The paclitaxel, etanidazole and paclitaxel+etanidazole nanoparticles effectively sensitized hypoxic tumor cells to radiation, their radiosensitizing effects: etanidazole-loaded nanoparticles < paclitaxel-loaded nanoparticles<paclitaxel+etanidazole nanoparticles.CONCLUSIONS:1. The synergistic radiosensitizing effect of etanidazole and paclitaxel on hypoxic HeLa cells was observed. The study provided a new combination of radiosensitizers to radiotherapy.2. The nanoparticles containing etanidazole / paclitaxel were prepared. Characterization of nanoparticle delivery system is satisfactory. The tumor cells swallowed the nanoparticles. The released drugs retained its bioactivity. Under this experimental condition, the radiosensitizing effects of drug-loaded nanoparticles were more significant than that of the free drugs. The radiosensitization produced by paclitaxel+etanidazole nanoparticles was more significant than that of the single drug-loaded nanoparticles. The drug-loaded nanoparticles have advantages in dosage form. This study provided a new idea for clinical application of radiosensitizers.更多还原