新型超声造影剂的制备和应用研究
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摘要
超声造影剂是一种可以显著增强医学超声诊断信号的辅助诊断试剂,提高超声诊断的敏感性和特异性,在医学影像诊断方面显示出良好的应用前景。目前关于超声造影剂的研究主要集中于两个方向:一是制备性能优良的新型造影剂;二是改进现有造影剂使其具有新功能。本文主要进行了基于表面活性剂的新型微泡超声造影剂的开发工作,以及在现有造影剂基础上制备多功能诊断试剂的研究工作。
本文通过声振空化法成功的得到了一种基于表面活性剂Span 60和Myrj 52的新型微泡超声造影剂——SM65。制备这种造影剂的材料均具有良好的生物相容性,无毒、无刺激、无致癌作用。SM65微泡造影剂浓度为(5.58±0.15)×109/mL,平均粒径为1.63±1.68μm,其中超过98.4%的微泡直径在7μm以下,符合作为临床超声造影剂的浓度和尺寸要求。此造影剂动物体内造影效果良好,平均增强时间超过5 min,满足临床诊断要求。此外,我们通过Langmuir单分子膜法研究了微泡膜层中分子的组成比例和稳定性原理,并推断了其内在稳定机制。结果显示Span 60分子和Myrj 52分子以摩尔比9:1的比例可以形成稳定的SM65微泡膜层。
在发展多功能诊断试剂的工作中,我们采用层层自组装法将碲化镉量子点与ST68微泡复合在一起,得到了具有荧光-超声双模式成像功能的新型复合诊断剂。荧光光谱和激光共聚焦显微镜的结果证实了量子点成功组装到了微泡的外表面,同时没有破坏微泡的结构。量子点修饰之后微泡的粒径相对减小,但仍满足临床使用的要求。体外和体内超声成像结果表明,这种量子点-微泡复合物仍保持良好的造影增强能力。另外,体外实验证明量子点-微泡复合物可以通过超声靶向破坏微泡技术在特定部位释放量子点达到靶向组织荧光成像的目的。制备这种双功能复合成像剂所采用的关键技术——层层自组装是一种非常简便通用的技术,在今后的研究中可以将量子点替换为药物、基因等多种物质,得到多功能的复合诊断/治疗试剂,它们将会在生命科学研究和临床医学实践中发挥重要的作用。
Ultrasound contrast agent (UCA), which can significantly improve the imaging quality in ultrasonic diagnosis with high sensitivity and specificity,is a promising assistant agent in medical imaging. Nowadays the research of UCA is focused on two fields: (1) the development of a novel contrast agent with high imaging quality; (2) the improvement of the existent UCA to develop multi-functional agents. This thesis successfully developed a novel surfactant-based UCA and a bi-mode imaging agent.
A novel microbubble UCA was prepared from sonication of two surfactans Span 60 and Myrj 52, and named as SM65. The materials used to prepare SM65 had good biocompatibility and no toxicity, stimulation or carcinogenesis. SM65 had its concentration of (5.58±0.15)×10 9/mL, and average size of 1.63±1.68μm with more than 98.4% of microbubbles smaller than 7μm, which met the requirement as UCA. In vivo ultrasonography confirmed the novel UCA had good contrast imaging quality and could provide contrast enhancement more than 5 min. Moreover, the Langmuir monolayer experiments gave both theoretical and practical explanation of the stability principles of SM65 microbubbles. The results showed that the surfactants Span 60 and Myrj 52 with a 9:1 molar ratio could make a stable SM65 shell.
On the other hand, a novel bi-mode diagnostic agent was fabricated in combination of ST68 microbubbles (MBs) and cadmium telluride quantum dots (CdTe QDs) by layer-by-layer (LbL) assembly technique. The photoluminescence experiment and confocal laser scanning microscopy confirmed that CdTe nano-particles were successfully deposited on the outer surface of the MBs. The static light scattering measurements showed that size distributions of MBs before and after QD adsorption met the size requirements for clinical application. Besides, the in vitro and in vivo ultrasound imaging indicated that the QD-modified MBs still maintained good contrast enhancement properties as ST68 MBs. Finally, the in vitro ultrasound-targeted microbubble destruction (UTMD) experiment of the QD-MB composites was carried out to validate the ability of MBs to deliver QDs for fluorescent imaging. The results showed that the QD-modified MBs not only maintained the capability of ultrasound imaging, but also could be used as targeted drug controlled-release system to deliver the QDs for cell and tissue fluorescent imaging by UTMD.
本文通过声振空化法成功的得到了一种基于表面活性剂Span 60和Myrj 52的新型微泡超声造影剂——SM65。制备这种造影剂的材料均具有良好的生物相容性,无毒、无刺激、无致癌作用。SM65微泡造影剂浓度为(5.58±0.15)×109/mL,平均粒径为1.63±1.68μm,其中超过98.4%的微泡直径在7μm以下,符合作为临床超声造影剂的浓度和尺寸要求。此造影剂动物体内造影效果良好,平均增强时间超过5 min,满足临床诊断要求。此外,我们通过Langmuir单分子膜法研究了微泡膜层中分子的组成比例和稳定性原理,并推断了其内在稳定机制。结果显示Span 60分子和Myrj 52分子以摩尔比9:1的比例可以形成稳定的SM65微泡膜层。
在发展多功能诊断试剂的工作中,我们采用层层自组装法将碲化镉量子点与ST68微泡复合在一起,得到了具有荧光-超声双模式成像功能的新型复合诊断剂。荧光光谱和激光共聚焦显微镜的结果证实了量子点成功组装到了微泡的外表面,同时没有破坏微泡的结构。量子点修饰之后微泡的粒径相对减小,但仍满足临床使用的要求。体外和体内超声成像结果表明,这种量子点-微泡复合物仍保持良好的造影增强能力。另外,体外实验证明量子点-微泡复合物可以通过超声靶向破坏微泡技术在特定部位释放量子点达到靶向组织荧光成像的目的。制备这种双功能复合成像剂所采用的关键技术——层层自组装是一种非常简便通用的技术,在今后的研究中可以将量子点替换为药物、基因等多种物质,得到多功能的复合诊断/治疗试剂,它们将会在生命科学研究和临床医学实践中发挥重要的作用。
Ultrasound contrast agent (UCA), which can significantly improve the imaging quality in ultrasonic diagnosis with high sensitivity and specificity,is a promising assistant agent in medical imaging. Nowadays the research of UCA is focused on two fields: (1) the development of a novel contrast agent with high imaging quality; (2) the improvement of the existent UCA to develop multi-functional agents. This thesis successfully developed a novel surfactant-based UCA and a bi-mode imaging agent.
A novel microbubble UCA was prepared from sonication of two surfactans Span 60 and Myrj 52, and named as SM65. The materials used to prepare SM65 had good biocompatibility and no toxicity, stimulation or carcinogenesis. SM65 had its concentration of (5.58±0.15)×10 9/mL, and average size of 1.63±1.68μm with more than 98.4% of microbubbles smaller than 7μm, which met the requirement as UCA. In vivo ultrasonography confirmed the novel UCA had good contrast imaging quality and could provide contrast enhancement more than 5 min. Moreover, the Langmuir monolayer experiments gave both theoretical and practical explanation of the stability principles of SM65 microbubbles. The results showed that the surfactants Span 60 and Myrj 52 with a 9:1 molar ratio could make a stable SM65 shell.
On the other hand, a novel bi-mode diagnostic agent was fabricated in combination of ST68 microbubbles (MBs) and cadmium telluride quantum dots (CdTe QDs) by layer-by-layer (LbL) assembly technique. The photoluminescence experiment and confocal laser scanning microscopy confirmed that CdTe nano-particles were successfully deposited on the outer surface of the MBs. The static light scattering measurements showed that size distributions of MBs before and after QD adsorption met the size requirements for clinical application. Besides, the in vitro and in vivo ultrasound imaging indicated that the QD-modified MBs still maintained good contrast enhancement properties as ST68 MBs. Finally, the in vitro ultrasound-targeted microbubble destruction (UTMD) experiment of the QD-MB composites was carried out to validate the ability of MBs to deliver QDs for fluorescent imaging. The results showed that the QD-modified MBs not only maintained the capability of ultrasound imaging, but also could be used as targeted drug controlled-release system to deliver the QDs for cell and tissue fluorescent imaging by UTMD.
引文
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