基于CNTs载药性和靶向性的新型超声造影剂的研究

发布时间：2018-11-15 17:22

【摘要】：目的基于碳纳米管载药性和靶向性设计制备一种集靶向、化疗于一体的新型多功能超声造影剂。通过研究制备复合微泡的工艺以及微泡中碳纳米管和紫杉醇的负载率,探索其体外抗肿瘤活性、体内外超声显像效果以及体内急性毒性;系统地对复合微泡进行临床前研究,为这种新型多功能超声造影剂的临床应用提供了基础研究数据。方法(1)采用声振空化法,以司盘-聚乙二醇(Span-PEG)为膜材,加入叶酸-碳纳米管-紫杉醇(folate-carbon nanotube-paclitaxel,FA-CNTs-PTX)复合物,制备复合FA-CNTs-PTX的司盘-聚乙二醇(Span-PEG)微泡超声造影剂;通过红外光谱仪、紫外分光光度计、热重仪、扫描电镜、透射电镜及激光粒度分析仪等对微泡结构和形貌进行表征分析。(2)采用多普勒超声诊断仪,分别考察复合微泡体外和体内超声造影效果。(3)选取乳腺癌MCF-7细胞、肝癌HepG2细胞和正常人脐静脉内皮HUVEC细胞作为试验模型,采用CCK-8试剂盒及AO/EB试剂盒双染色法检测复合微泡超声造影剂体外抗肿瘤活性。通过流式细胞术定量分析复合微泡对乳腺癌MCF-7细胞和正常人脐静脉内皮HUVEC细胞的凋亡率。(4)以昆明小鼠为模型,通过急性毒性试验确定复合微泡超声造影剂对小鼠的最大耐受量。结果(1)所制备的复合FA-CNTs-PTX的Span-PEG微泡表面光滑、平均粒径为442 nm,微泡中碳纳米管和紫杉醇的负载率分别为1.69%和47.9%。成功将FA-CNTsPTX靶向载药复合物包覆于Span-PEG微泡中,微泡为空心球体,粒径分布均匀且为纳米级别。(2)与生理盐水和Span-PEG微泡相比,复合FA-CNTs-PTX微泡超声造影剂在体外和体内均具有更显著的增强超声信号的性质,并且超声成像的清晰度有所提高。(3)复合FA-CNTs-PTX的微泡超声造影剂对乳腺癌MCF-7细胞有明显的增殖抑制作用,且主要取决于较高的载药率及微泡的纳米尺寸;复合FA-CNTs-PTX的微泡超声造影剂能够影响乳腺癌MCF-7细胞的增殖抑制率及细胞的凋亡周期,并对细胞MCF-7的增殖抑制作用程度高于肝癌HepG2细胞;复合FA-CNTs-PTX的微泡超声造影剂对乳腺癌MCF-7细胞的早期凋亡率34.85%远大于对正常人脐静脉内皮HUVEC细胞的早期凋亡率14.99%,复合FA-CNTs-PTX的微泡超声造影剂对乳腺癌MCF-7细胞具有明显的靶向抑制作用。(4)最大耐受剂量下昆明小鼠没有出现死亡及不良反应,最大耐受剂量为50 kg成人的350倍。结论本研究设计和制备的复合FA-CNTs-PTX微泡超声造影剂有望成为一种良好的集造影和靶向治疗为一体的超声造影剂。首先,复合微泡的结构满足作为造影剂的理论条件,在体内外均具有理想的增强超声显影功能;其次,该复合微泡成功负载紫杉醇药物,实现靶向抑制乳腺癌MCF-7细胞增殖作用。最后,复合微泡作用于昆明小鼠的最大耐受剂量已经远高于成人用量的百倍,所以复合FA-CNTs-PTX微泡超声造影剂在体内安全稳定,且提供比较理想的超声显影效果,是一种高效的新辅助化疗试剂,为下一步体内递药提供研究的药理模型。
[Abstract]:Objective To prepare a novel multi-functional ultrasound contrast agent integrated with target and chemotherapy based on the properties of carbon nanotubes and the targeted design. The method for preparing the composite micro-bubble and the loading rate of the carbon nano-tube and the paclitaxel in the micro-bubble are researched, the in vitro anti-tumor activity, the in-vivo ultrasound imaging effect and the in-vivo acute toxicity are explored, The basic research data is provided for the clinical application of the novel multifunctional ultrasound contrast agent. The method comprises the following steps: (1) adopting an acoustic vibration cavitation method, taking the pan-polyethylene glycol (Span-PEG) as a membrane material, adding a folic acid-carbon nano-tube-paclitaxel (FA-CNTs-PTX) complex, and preparing a Si-polyethylene glycol (Span-PEG) micro-bubble ultrasonic contrast agent of the composite FA-CNTs-PTX; and through an infrared spectrometer, The micro-bubble structure and morphology were characterized and analyzed by ultraviolet spectrophotometer, thermogravimeter, scanning electron microscope, transmission electron microscope and laser particle size analyzer. (2) Doppler ultrasonic diagnostic apparatus was used to study the effect of combined microvesicle in vitro and in vivo. (3) The anti-tumor activity of human breast cancer MCF-7 cells, liver cancer HepG2 cells and human umbilical vein endothelial HUVEC cells was selected as the test model, and the anti-tumor activity of the composite micro-bubble ultrasound contrast agent was detected by using the CCK-8 kit and the AO/ EB kit double staining method. The apoptosis rate of HUVEC cells in human umbilical vein endothelial cells (MCF-7) and human umbilical vein endothelial cells (HUVEC) was analyzed by flow cytometry. (4) The maximum tolerance of the compound micro-bubble ultrasound contrast agent to the mice was determined by acute toxicity test with Kunming mice as the model. Results (1) The surface of the Span-PEG microfoam of the composite FA-CNTs-PTX was smooth, the average particle size was 442 nm, and the loading rates of the carbon nanotubes and the paclitaxel in the micro-bubbles were 1.69% and 44.9%, respectively. The FA-CNTPTX-targeting drug-containing compound was successfully coated in the Span-PEG micro-bubble, and the micro-bubble is a hollow sphere, and the particle size distribution is uniform and the nanometer level is nanometer. (2) Compared with the normal saline and the Span-PEG microvesicle, the composite FA-CNTs-PTX micro-bubble ultrasound contrast agent has more significant enhancement of the nature of the ultrasound signal both in vitro and in vivo, and the clarity of the ultrasound imaging is improved. and (3) the micro-bubble ultrasound contrast agent of the compound FA-CNTs-PTX has obvious proliferation inhibition effect on the breast cancer MCF-7 cells, and mainly depends on the higher drug loading rate and the nano size of the micro-bubbles; and the micro-bubble ultrasound contrast agent of the composite FA-CNTs-PTX can influence the proliferation inhibition rate of the breast cancer MCF-7 cells and the apoptosis cycle of the cells, The inhibition of the proliferation of the cell line MCF-7 was higher than that of the HepG2 cells. The early apoptotic rate of the composite FA-CNTs-PTX was 34. 85%, which was much higher than that of the human umbilical vein endothelial HUVEC cells. The micro-bubble ultrasound contrast agent of compound FA-CNTs-PTX has obvious targeted inhibition on breast cancer MCF-7 cells. (4) No death and adverse reaction occurred in Kunming mice at the maximum tolerated dose, and the maximum tolerated dose was 350 times of that of 50 kg of adult. Conclusion The complex FA-CNTs-PTX micro-bubble ultrasound contrast agent designed and prepared in this study is expected to be a good contrast agent for both contrast and targeted therapy. firstly, the structure of the composite micro-bubble meets the theoretical condition as a contrast agent, and has an ideal enhanced ultrasonic developing function in the outside of the body; secondly, the composite micro-bubble is successfully loaded with the paclitaxel medicament to realize the targeted inhibition of the proliferation of the breast cancer MCF-7 cell. and finally, the maximum tolerated dose of the composite micro-bubble on the Kunming mouse is much higher than that of the adult, so the composite FA-CNTs-PTX micro-bubble ultrasonic contrast agent is safe and stable in the body, The pharmacological model of the study was provided for the next in-vivo delivery.
【学位授予单位】：佳木斯大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943

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