高强度聚焦超声诱导阿苯达唑热敏脂质体杀伤细粒棘球蚴的研究

发布时间：2018-04-18 16:47

本文选题：高强度聚焦超声 + 阿苯达唑　；参考：《重庆医科大学》2015年硕士论文

【摘要】：目的探索高强度聚焦超声(High intensity focused ultrasound, HIFU)诱导阿苯达唑热敏脂质体(Thermosensitive liposomes loaded with albendazole, TSL-ABZ)杀伤包虫原头蚴的效果。利用HIFU的热效应来诱导热敏脂质体靶向释药,从而提高阿苯达唑的生物利用度,增强对包虫病的治疗效果。方法1.采用单因素实验,来确定阿苯达唑热敏脂质体的制备方法。通过考察有机溶剂种类及用量、合成磷脂间的比例、水合介质pH值、脂类与药物比例、水浴温度等因素,初步确定制剂处方和工艺条件。2.通过透射电镜、Nano ZS90Zetasizer粒径测量、傅里叶近红外光谱分析、热重分析、体外释放浓度测试等手段对该药物载体进行表征考察,以证明是否成功制备阿苯达唑热敏脂质体。3.观察阿苯达唑热敏脂质体对离体原头蚴的影响。在不同水浴温度下,将不同比例的阿苯达唑热敏脂质体加入含有原头蚴的囊液中,测定药物浓度及在制剂作用影响下原头蚴的活力。4.观察HIFU诱导阿苯达唑热敏脂质体对离体原头蚴的杀伤效果。在一定高强度超声剂量下,将不同比例的制剂药物加入原头蚴悬液中,在光镜下观察形态改变并计数其死亡率,HE染色、透射电镜观察原头蚴组织病理结构的变化,TUNEL荧光染色观察原头蚴核凋亡情况。结果1.实验筛选出最佳的制备处方为：在药脂比为1：40,DPPC:MPPC:DSPG:ABZ成分比为66：7：5：3,缓冲液pH值为5.3,超声时间为15 min,水浴温度为38℃条件下,热敏脂质体的平均包封率为78.7%,ABZ载药率为1.91%。2.透射电镜下空白热敏脂质体为环形空心结构,而阿苯达唑热敏脂质体呈黑色圆球状；粒径分析仪测定制备的TSL-ABZ的平均粒径为316 nm, PDI为0.533,Zeta电位为-9.84 mV；红外光谱测试表明药物与载体间未发生化学作用力,且药物ABZ的结构也未发生改变,并证明药物被包含在脂质体结构中；DSC曲线证实制备的脂质体具有热敏特性,其相变温度约为42.1℃。3.在水浴时间24 h,水浴温度为42℃时,TSL-ABZ 1000μl组能最大程度诱导原头蚴死亡,死亡率较其他组更高。4.经过温度测试后,选择达到相变温度的最适HIFU辐照剂量(75W12 s),各实验组原头蚴的死亡率呈现一定的增长趋势,尤以HIFU+TSL-ABZ 1000μl组为最高,形态学观察发现皮层破坏严重,钙颗粒消失,空泡化严重,头钩脱落,部分结构甚至消失。荧光染色可见大量凋亡的细胞核。结论1.通过本实验方法制备的热敏脂质体成功包裹了阿苯达唑标准品,脂质体的形貌规则且呈单分散状态。粒径也已达到纳米级,分散均一,制剂体系比较稳定。2.热敏性磷脂可以控制药物释放,可依据实验条件选择性的作用于实验对象。在室温和体温时,脂质体内的药物不释放,当温度超过相变辅料的熔点时,药物释放具有爆炸式的特点。3.阿苯达唑热敏脂质体在相变温度条件下释放药物,并对离体原头蚴有杀伤作用。4.HIFU可诱导阿苯达唑热敏脂质体释放,从而增强对离体原头蚴的杀伤效果。
[Abstract]:Objective to explore the high intensity focused ultrasound (High intensity, focused ultrasound, HIFU) induced by albendazole thermosensitive liposomes (Thermosensitive liposomes loaded with albendazole, TSL-ABZ) killing Echinococcus hydatid protoscolex effect. To induce thermosensitive liposome targeting drug release by the thermal effect of HIFU, so as to improve the albendazole bioavailability and to enhance the therapeutic effect of hydatid disease. Methods 1. single factor experiments to determine the preparation method of albendazole thermosensitive liposomes. Through the investigation of organic solvent type and amount, the ratio between the synthesis of phospholipids, water and pH value of medium, lipid and drug proportion factors, water bath temperature, preliminary determine prescription. And the process conditions of.2. by transmission electron microscopy, Nano ZS90Zetasizer particle size measurement, Fu Liye analysis of near infrared spectroscopy, thermogravimetric analysis, in vitro release concentration were characterized on the drug carrier, In order to prove whether the successful preparation of albendazole thermosensitive liposome.3. of albendazole thermosensitive liposomes in vitro of protoscolex. At different temperatures, different proportion of albendazole thermosensitive liposomes containing added cystic fluid protoscolex in test and observe protoscolex the activity of.4. in the preparation of HIFU under the action of impact induced by albendazole thermosensitive liposomes in vitro protoscolex the killing effect of the drug concentration. In some high intensity ultrasound doses, the drug adding different proportion of protoscolices in suspension, observe the morphological changes under light microscope and counting the dead rate of HE staining, observe the changes of pathological structure of protoscolex TEM observation of protoscolex nuclear apoptosis TUNEL staining. Results of the 1. experiments were selected as the best prescription preparation: the drug lipid ratio for 1:40, DPPC:MPPC:DSPG:ABZ component is 66:7:5:3, pH value of buffer 5.3, ultrasonic time was 15 min, water bath temperature of 38 DEG C, thermosensitive liposomes entrapment rate was 78.7%, the drug loading rate of 1.91%.2. ABZ transmission electron microscope blank thermosensitive liposome as a hollow structure, and albendazole thermosensitive liposomes were black spherical particle size analysis instrument; TSL-ABZ prepared by the average particle size is 316 nm, PDI 0.533, Zeta potential is -9.84 mV; infrared spectroscopy test showed that the drug with the carrier without the occurrence of chemical reaction and structure of drug ABZ has not changed, and that the drug contained in the liposome structure; DSC curve indicated that the prepared liposome with thermal characteristics, the phase transition temperature is about 42.1.3. in the water bath time is 24 h, the water bath temperature is 42 degrees centigrade, TSL-ABZ 1000 l group to the maximum extent induced protoscolex death, mortality higher than other groups.4. after the temperature test, at the phase transition temperature The optimum HIFU irradiation dose (75W12 s), the experimental group of protoscolex mortality showed a growth trend, especially in HIFU+TSL-ABZ 1000 l group was the highest, the morphological observation of cortical destruction, the calcium particles disappear, severe vacuolization, head hook off, part of the structure or even disappear. Fluorescent staining showed the apoptosis the conclusion of the 1. nucleus. By this method the preparation of thermosensitive liposomes successfully encapsulated albendazole standard, morphology of liposomes and monodispersed. The particle size has reached the nanometer level, uniform dispersion, preparation system is stable.2. heat sensitive phospholipids can control drug release, according to the experimental conditions of selective the effect on the subject. In room temperature and body temperature, body lipid drug release, when the temperature exceeds the melting point of phase change materials, drug release with the explosive characteristics of.3. albendazole thermosensitive liposomes in phase The release of drugs and killing of the original larvae in different temperature conditions can induce the release of albendazole thermosensitive liposomes, and enhance the killing effect of.4.HIFU on the isolated larvae.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R532.31

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