新型载药微泡的制备及体内初步实验研究

发布时间：2018-03-03 16:09

本文选题：PLGA纳米粒　切入点：脂质微泡　出处：《重庆医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：第一部分包裹紫杉醇的高分子纳米粒与脂质微泡复合体的制备研究目的利用生物素-亲和素技术制备一种包裹紫杉醇的新型载药微泡，并对其连接效果、理化性质进行检测。方法采用单乳化法制备包裹紫杉醇的PLGA-COOH纳米粒（Pac-PLGA），检测其包封率与载药量，采用碳二亚胺共价连接法将其与链霉亲和素相连，免疫荧光检测链霉亲和素与Pac-PLGA的连接情况。采用机械振荡法制备生物素化的脂质微泡（BIO-MB）及普通脂质微泡（MB）。连接分为2组：生物素-亲和素组（SA-Pac-PLGA+BIO-MB），对照组（Pac-PLGA+MB）。用激光共聚焦显微镜分别观察2组在不同时间点脂质微泡与Pac-PLGA的连接情况。结果Pac-PLGA的平均粒径为(131.1±29.7)nm，扫描电镜观察呈球形，大小均匀，分散度好。高效液相色谱法测得PLGA纳米粒对紫杉醇的包封率为(85±2.1)%，载药量为（4.25±0.16）%，荧光检测链霉亲和素成功地与Pac-PLGA连接。激光共聚焦观察生物素-亲和素组Pac-PLGA较多地连接在BIO-MB的表面。而对照组MB表面未见Pac-PLGA的聚集。结论本实验成功地制备出包裹紫杉醇的新型载药微泡，有望为恶性肿瘤的治疗提供一种理想的药物载体和靶向给药系统。第二部分包裹紫杉醇的高分子纳米粒与脂质微泡复合体在小鼠肝脏的定位释放研究目的探讨新型载药微泡在一定声强的超声定位辐照下，紫杉醇及PLGA纳米粒在小鼠肝脏的定位释放情况。方法利用生物素-亲和素技术制备包裹紫杉醇的新型载药微泡。将实验小鼠分为3组：载紫杉醇PLGA纳米粒组（Pac-PLGA纳米粒组）、载紫杉醇PLGA纳米粒与超声脂质微泡混合组（混合组）、包裹紫杉醇的新型载药微泡组，经尾静脉注入小鼠体内后，3组小鼠均用相同声强的超声经体表定点辐照小鼠肝脏1min，于处理1h后断颈处死取肝组织，用高效液相色谱法测定小鼠肝脏组织中紫杉醇的浓度，用激光共聚焦显微镜观察小鼠肝组织切片每高倍视野下（x400）荧光标记的Pac-PLGA纳米粒的数量。结果高效液相色谱法测得新型载药微泡组小鼠肝组织中的紫杉醇含量为（5.335土1.087）ug/g,高于混合组（4.067土0.954）ug/g和Pac-PLGA纳米粒组（2.908土0.925）ug/g,p均0.01，且混合组高于Pac-PLGA纳米粒组（p0.01）。激光共聚焦观察每高倍视野下（x400）荧光标记的Pac-PLGA纳米粒的数量，Pac-PLGA纳米粒组数量为(17.590土5.811)个，混合组数量为(31.227土7.177）个，新型载药微泡组数量为(47.500土7.301)个，，新型载药微泡组Pac-PLGA纳米粒数量高于其余各组（p0.01），且混合组高于Pac-PLGA纳米粒组（p0.01）。结论新型载药微泡组小鼠肝组织中的紫杉醇含量及PLGA纳米粒的数量明显高于其余各组。新型载药微泡能够提高靶组织区内的药物浓度及PLGA纳米粒的数量，从而提高疾病的治疗效果。
[Abstract]:Preparation of macromolecular nanoparticles and lipid microvesicles coated with paclitaxel. Aim to prepare a novel paclitaxel encapsulated microbubble by biotin-avidin technique, and to determine its binding effect and physicochemical properties. Methods Pac-PLGA, a paclitaxel coated PLGA-COOH nanoparticles, was prepared by single emulsification method. The encapsulation efficiency and drug loading of paclitaxel were determined. The carbodiimide covalent bonding method was used to bind paclitaxel to streptavidin. The connection between streptavidin and Pac-PLGA was detected by immunofluorescence. Biotin lipid microbubbles BIO-MBs and ordinary lipid microbubbles were prepared by mechanical oscillation method. The connections were divided into two groups: biotin group, SA-Pac-PLGA BIO-MBN group, control group, pac-PLGA MBM. The connection between lipid microbubbles and Pac-PLGA at different time points was observed by light confocal microscope. Results the average diameter of Pac-PLGA was 131.1 卤29.7nm.The Pac-PLGA was spherical and uniform in size. High performance liquid chromatography (HPLC) showed that the encapsulation efficiency of PLGA nanoparticles to paclitaxel was 85 卤2.1 and the drug load was 4.25 卤0.16.The fluorescent detection of Streptomycin was successfully connected to Pac-PLGA. Laser confocal observation showed that Pac-PLGA in biotin / avidin group was more concatenated. The Pac-PLGA aggregation was not observed on the surface of BIO-MB in the control group. Conclusion the microbubbles coated with paclitaxel were successfully prepared in this experiment, which is expected to provide an ideal drug carrier and targeted drug delivery system for the treatment of malignant tumors. The second part: localization and release of paclitaxel encapsulated polymer nanoparticles and lipid microvesicles in mouse liver. Objective to investigate the localization release of paclitaxel and PLGA nanoparticles in the liver of mice. Methods A novel paclitaxel coated microbubble was prepared by biotin avidin technique. The experimental mice were divided into three groups: pac-PLGA nanoparticles group, pac-PLGA nanoparticles group, paclitaxel-loaded PLGA nanoparticles mixed with ultrasonic lipid microbubbles, the experimental mice were divided into three groups: paclitaxel loaded PLGA nanoparticles group, pac-PLGA nanoparticles group, paclitaxel loaded PLGA nanoparticles and ultrasonic lipid microbubbles. Group A (mixed group, paclitaxel coated microbubble group), After injected into the tail vein of mice, the liver of all the three groups were irradiated with the same sound intensity ultrasound at the fixed point of the body surface for 1 min. After 1 hour of treatment, the liver tissues were killed and the concentration of paclitaxel in the liver tissue of the mice was determined by high performance liquid chromatography (HPLC). The number of Pac-PLGA nanoparticles labeled with X 400) in mouse liver tissue sections was observed by laser confocal microscopy. Results the content of paclitaxel in the liver tissue of mice in the new drug loaded microbubble group was determined by HPLC, which was higher than that in the mixed group (4.067 卤0.954g / g) and the Pac-PLGA nanoparticles group (0.925g / g / g), and the content of paclitaxel in the liver tissue of the mixed group was higher than that of the Pac-PLGA nano-particle group (p 0.01g), and the content of paclitaxel was higher in the mixed group than that in the Pac-PLGA nanoparticles group (p 0.01). The number of Pac-PLGA nanoparticles labeled with fluorescence in each high-power field of vision was 17.590 卤5.811, and the number of Pac-PLGA nanoparticles was 5.811. The number of mixed group was 31.227 卤7.177, the number of new drug loaded microbubble group was 47.500 卤7.301), the number of Pac-PLGA nanoparticles in new drug loaded microbubble group was higher than that of other groups, and the mixing group was higher than Pac-PLGA group. Conclusion the contents of paclitaxel and the number of PLGA nanoparticles in liver tissue of the new drug loaded microbubble group were significantly higher than those in the other groups, and the new drug loaded microbubbles could increase the concentration of drug and the number of PLGA nanoparticles in the target tissue. So as to improve the therapeutic effect of the disease.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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