新型微泡超声造影剂的制备及性能与应用的研究
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摘要
超声诊断作为一种无创、低成本、实时的成像技术,是目前应用最为广泛的成像方法。然而,由于传统超声诊断技术的敏感性和分辨率都较低,限制了它的应用。微泡超声造影剂(Ultrasound Contrast Agent,UCA)因具有较强的回波反射性能,能够显著增强医学超声检测信号。在诊断方面,它能提高诊断的敏感性和特异性,在炎症显像、血栓显像以及癌症的早期诊断方面应用十分广泛。除了作为诊断药剂外,超声造影剂在分子影像、促进血栓溶解、基因转染和药物运输定位释放等多个领域都具有重要的研究和应用价值。本课题针对超声医学领域的需求,开发具有良好超声显影特性的新型微泡超声造影剂。
针对目前制备的微泡超声造影剂稳定性欠佳的问题,制备了聚电解质膜包覆的微泡超声造影剂。首先,采用声振空化的方法制备了表面带负电荷的ST68微泡。然后以该微泡为模板,以带相反电荷和具有良好生物相容性的聚电解质为包膜材料,采用静电层层自组装的方法成功制备了聚电解质膜包覆的微泡超声造影剂。得到的微泡分散性好,粒度分布均匀。体内外超声造影结果表明,制得的微泡超声造影剂仍能维持其良好的超声造影效果。在前面工作的基础上,进一步制备了泡壁含有磁性纳米粒子的磁性微泡。首先,采用多元醇法制备了表面带负电荷的磁性纳米粒子,而后采用层层自组装的方法,通过聚乙烯亚胺和磁性纳米粒子在微泡表面的交替沉积,制得了具有超声/磁场双重响应特性的微泡超声造影剂。制得的磁性微泡形态规整,粒度分布均匀,在磁场中具有良好的响应特性。组装磁性纳米粒子后微泡的形态和粒径较组装前没有发生明显变化。体内外超声造影结果表明磁性微泡具有良好的超声造影效果。此外,我们在体外成功地实现了超声监测下的磁靶向,为今后进一步研究可满足诊断和治疗双重作用的磁靶向微泡超声造影剂打下了良好的研究基础。
在总结前人工作的基础上,我们从分子结构出发,选择合适的成膜材料,制备了新型基于表面活性剂的微泡超声造影剂。新型微泡以司班60(Span 60)和聚乙二醇单硬脂酸酯(PEG40S)为成膜材料,全氟丙烷(C3F8)为包封气体,采用声振空化的方法制备而成。制得的微泡粒度分布均匀,分散性好,平均粒径约为2.08±1.27μm,超过99%的微泡直径小于8μm,表明它们尺寸适宜,可以作为超声造影剂用于静脉注射。体内外超声造影结果表明,新型微泡在脉冲反向谐波和能量多普勒模式下均具有很好的显影增强效果,造影过程中兔基本生命体征未见明显变化。在制得了微米级气泡的基础上,采用离心的方法从母液微泡悬液中分离得到了尺寸可控的纳米级气泡。动物体内能量多普勒模式造影结果表明,它可以显著增强兔肾脏能量多普勒信号。为了了解制得的新型微泡的结构和稳定机制,采用Langmuir单分子膜技术在分子水平上考察了微泡成膜组分的单分子膜行为。结果表明,Span 60能形成低表面张力的壳层,有效地降低了腔内气体向外部的弥散;PEG40S分子的加入构建了一个亚能垒,阻止了微泡间的凝聚融合,从而赋予了其良好的稳定性。将微泡与不同比例的混合单分子膜的π-A等温曲线进行比较,推测微泡膜层中Span 60与PEG40S的分子数比例大约为9:1。
以生物相容性和安全性俱佳的可降解高分子聚合物聚乳酸(PLA)为成膜材料,量子点(QDs)为发光物质,采用双乳溶剂挥发法和冷冻干燥技术相结合制备具有超声/荧光双模式显影功能的高分子材料微泡——QDs@PLA微泡。制得的微泡呈规则的球形,具有中空结构,粒度分布均匀,能够满足超声造影剂对尺寸的基本要求。荧光发射光谱和荧光显微镜结果表明,QDs@PLA微泡能维持QDs良好的发光特性,PLA壳层有效地增加了QDs的荧光稳定性。体内外超声造影结果表明,QDs@PLA微泡具有良好的回声特性。体内外荧光成像结果表明,QDs@PLA微泡具有很好的荧光成像能力,与超声造影结果具有同步性。
As a non-invasive, low cost and real-time imaging modality, ultrasonic diagnosis is the most widely used imaging method at present. However, due to the relatively low sensitivity and resolution of traditional ultrasonic diagnosis, its further applications are limited. Microbubble ultrasound contrast agent can significantly improve the signal to noise ration of the medical ultrasound because of the strong echo reflection of microbubbles. In diagnosis, the advent of ultrasound contrast agent promises to improve the sensitivity and specificity of ultrasound in the detection of inflammation, thrombosis and early cancer. Besides working as a diagnostic agent, ultrasound contrast agent has been extensively studied in molecular imaging, promoting thrombolysis and gene transfection and site-specific drug delivery and release, etc. This dissertation is focus on the needs of ultrasound medicine, aiming to develop novel microbubble ultrasound contrast agent with excellent ultrasonic contrast imaging characteristics.
In view of the instability of the present microbubble ultrasound contrast agent, polyelectrolyte multilayer film-coated microbubbles were developed. Firstly, ST68 microbubbles with negative charge were prepared by acoustic cavitation method. Subsequently, the resulting microbubbles were coated with oppositely charged and good biocompatibility polyelectrolytes by microbubble-templated layer-by-layer self-assembly technique via electrostatic interaction. The obtained microbubbles had a good dispersion and their size distribution is relative uniform. In vitro and in vivo ultrasound imaging results indicated that the polyelectrolyte multilayer film-coated microbubble ultrasound contrast agent could still maintain the good performance of initial microbubbles. On the basis of precedent work, we fabricated magnetic microbubbles which contained magnetic nanoparticles on the surface of microbubbles. Firstly, magnetic nanoparticles with negative charge were synthesized by polyol process. Then, the ultrasound/magnetic field dual response microbubble ultrasound contrast agent was fabricated through alternately depositing polyethylenimine and magnetic nanoparticles onto microbubbles by layer-by-layer self-assembly technique. The obtained magnetic microbubbles appeared uniformly with a tight size distribution and were provided with good response in magnetic field. The configuration and diameter of microbubbles after assembling magnetic nanoparticles were almost the same as those of non-assembled ones. In vitro and in vivo ultrasound contrast effects indicated that the magnetic microbubbles had good ultrasound imaging performance. In addition, we succeed in magnetic targeting in vitro under ultrasonic monitoring. It will serve a double purpose for diagnosis and treatment, which would establish well foundation for further magnetic targeting microbubble ultrasound contrast agent.
Based on the work of the formers, we proceed from molecular structure, selecting suitable microbubble forming materials, to prepare novel microbubble ultrasound contrast agent based on surfactants. The novel perfluoropropane-containing microbubbles were fabricated using ultrasonication of a sorbitan monostearate (Span 60) and polyoxyethylene 40 stearate (PEG40S). The obtained microbubbles distributed uniformly with an average diameter of 2.08±1.27μm. More than 99% of the microbubbles had a mean diameter less than 8μm, indicating that they were appropriately sized for intravenous administration as ultrasound contrast agent. The preliminary in vitro and in vivo ultrasound imaging study showed that such novel microbubbles demonstrated excellent enhancement under grey-scale pulse inversion harmonic imaging and power Doppler imaging. There were no abnormal changes of the rabbit’s basic life signs during the study. Based on the facts that we have fabricated micro-scale microbubbles, a nano-sized population with controlled mean diameter could be sorted by using centrifugal classifier from the parent polydisperse suspensions. Preliminary study with the nano-scale bubbles in vivo showed that such nanobubbles provided an excellent power Doppler enhancement. In order to understand the structure and stabilization mechanism of the novel microbubbles, Langmuir monolayer technique was performed to investigate the monolayer behaviour of microbubble forming components at molecular level. The results indicated that Span 60 could form a low surface tension monolayer which could diminish the gas diffusion from the core into the aqueous medium. The incorporation of PEG40S into the monolayer shell created a sub-energy barrier to prevent aggregation among microbubbles which impart good stability to the microbubbles. Based on the comparison ofπ-A isotherms for microbubbles and those obtained for mixtures of surfactants at different molar ratios, we suggested a composition in the microbubble skin close to nine molecules of Span 60 to each molecule of PEG40S. Polymeric microbubble ultrasound contrast agent (QDs@PLA microbubbles) with ultrasound/fluorescence bi-mode imaging functions was fabricated using double emulsion solvent evaporation and lyophilization methods. Polylactide (PLA), a biocompatible, safety and biodegradable high molecular polymer, was employed as the shell material and quantum dots (QDs) were employed as the luminescent material. The morphology of the polymeric microbubbles observed with scanning electron microscope appeared regular spherical with hollow structure and have a tight size distribution, which could meet the requirement of size for ultrasound contrast agent. The results of fluorescence emission spectrum and fluorescence microscope indicated that QDs@PLA microbubbles could maintain the good luminescence properties of QDs and PLA microbubble shell impart good fluorescence stability to the QDs. In vitro and in vivo ultrasound contrast effects indicated that QDs@PLA microbubbles showed high echogenicity. The fluorescence imaging in vitro and in vivo showed that QDs@PLA microbubbles exhibited excellent fluorescence imaging ability, and were in synchronism with ultrasound contrast effect.
针对目前制备的微泡超声造影剂稳定性欠佳的问题,制备了聚电解质膜包覆的微泡超声造影剂。首先,采用声振空化的方法制备了表面带负电荷的ST68微泡。然后以该微泡为模板,以带相反电荷和具有良好生物相容性的聚电解质为包膜材料,采用静电层层自组装的方法成功制备了聚电解质膜包覆的微泡超声造影剂。得到的微泡分散性好,粒度分布均匀。体内外超声造影结果表明,制得的微泡超声造影剂仍能维持其良好的超声造影效果。在前面工作的基础上,进一步制备了泡壁含有磁性纳米粒子的磁性微泡。首先,采用多元醇法制备了表面带负电荷的磁性纳米粒子,而后采用层层自组装的方法,通过聚乙烯亚胺和磁性纳米粒子在微泡表面的交替沉积,制得了具有超声/磁场双重响应特性的微泡超声造影剂。制得的磁性微泡形态规整,粒度分布均匀,在磁场中具有良好的响应特性。组装磁性纳米粒子后微泡的形态和粒径较组装前没有发生明显变化。体内外超声造影结果表明磁性微泡具有良好的超声造影效果。此外,我们在体外成功地实现了超声监测下的磁靶向,为今后进一步研究可满足诊断和治疗双重作用的磁靶向微泡超声造影剂打下了良好的研究基础。
在总结前人工作的基础上,我们从分子结构出发,选择合适的成膜材料,制备了新型基于表面活性剂的微泡超声造影剂。新型微泡以司班60(Span 60)和聚乙二醇单硬脂酸酯(PEG40S)为成膜材料,全氟丙烷(C3F8)为包封气体,采用声振空化的方法制备而成。制得的微泡粒度分布均匀,分散性好,平均粒径约为2.08±1.27μm,超过99%的微泡直径小于8μm,表明它们尺寸适宜,可以作为超声造影剂用于静脉注射。体内外超声造影结果表明,新型微泡在脉冲反向谐波和能量多普勒模式下均具有很好的显影增强效果,造影过程中兔基本生命体征未见明显变化。在制得了微米级气泡的基础上,采用离心的方法从母液微泡悬液中分离得到了尺寸可控的纳米级气泡。动物体内能量多普勒模式造影结果表明,它可以显著增强兔肾脏能量多普勒信号。为了了解制得的新型微泡的结构和稳定机制,采用Langmuir单分子膜技术在分子水平上考察了微泡成膜组分的单分子膜行为。结果表明,Span 60能形成低表面张力的壳层,有效地降低了腔内气体向外部的弥散;PEG40S分子的加入构建了一个亚能垒,阻止了微泡间的凝聚融合,从而赋予了其良好的稳定性。将微泡与不同比例的混合单分子膜的π-A等温曲线进行比较,推测微泡膜层中Span 60与PEG40S的分子数比例大约为9:1。
以生物相容性和安全性俱佳的可降解高分子聚合物聚乳酸(PLA)为成膜材料,量子点(QDs)为发光物质,采用双乳溶剂挥发法和冷冻干燥技术相结合制备具有超声/荧光双模式显影功能的高分子材料微泡——QDs@PLA微泡。制得的微泡呈规则的球形,具有中空结构,粒度分布均匀,能够满足超声造影剂对尺寸的基本要求。荧光发射光谱和荧光显微镜结果表明,QDs@PLA微泡能维持QDs良好的发光特性,PLA壳层有效地增加了QDs的荧光稳定性。体内外超声造影结果表明,QDs@PLA微泡具有良好的回声特性。体内外荧光成像结果表明,QDs@PLA微泡具有很好的荧光成像能力,与超声造影结果具有同步性。
As a non-invasive, low cost and real-time imaging modality, ultrasonic diagnosis is the most widely used imaging method at present. However, due to the relatively low sensitivity and resolution of traditional ultrasonic diagnosis, its further applications are limited. Microbubble ultrasound contrast agent can significantly improve the signal to noise ration of the medical ultrasound because of the strong echo reflection of microbubbles. In diagnosis, the advent of ultrasound contrast agent promises to improve the sensitivity and specificity of ultrasound in the detection of inflammation, thrombosis and early cancer. Besides working as a diagnostic agent, ultrasound contrast agent has been extensively studied in molecular imaging, promoting thrombolysis and gene transfection and site-specific drug delivery and release, etc. This dissertation is focus on the needs of ultrasound medicine, aiming to develop novel microbubble ultrasound contrast agent with excellent ultrasonic contrast imaging characteristics.
In view of the instability of the present microbubble ultrasound contrast agent, polyelectrolyte multilayer film-coated microbubbles were developed. Firstly, ST68 microbubbles with negative charge were prepared by acoustic cavitation method. Subsequently, the resulting microbubbles were coated with oppositely charged and good biocompatibility polyelectrolytes by microbubble-templated layer-by-layer self-assembly technique via electrostatic interaction. The obtained microbubbles had a good dispersion and their size distribution is relative uniform. In vitro and in vivo ultrasound imaging results indicated that the polyelectrolyte multilayer film-coated microbubble ultrasound contrast agent could still maintain the good performance of initial microbubbles. On the basis of precedent work, we fabricated magnetic microbubbles which contained magnetic nanoparticles on the surface of microbubbles. Firstly, magnetic nanoparticles with negative charge were synthesized by polyol process. Then, the ultrasound/magnetic field dual response microbubble ultrasound contrast agent was fabricated through alternately depositing polyethylenimine and magnetic nanoparticles onto microbubbles by layer-by-layer self-assembly technique. The obtained magnetic microbubbles appeared uniformly with a tight size distribution and were provided with good response in magnetic field. The configuration and diameter of microbubbles after assembling magnetic nanoparticles were almost the same as those of non-assembled ones. In vitro and in vivo ultrasound contrast effects indicated that the magnetic microbubbles had good ultrasound imaging performance. In addition, we succeed in magnetic targeting in vitro under ultrasonic monitoring. It will serve a double purpose for diagnosis and treatment, which would establish well foundation for further magnetic targeting microbubble ultrasound contrast agent.
Based on the work of the formers, we proceed from molecular structure, selecting suitable microbubble forming materials, to prepare novel microbubble ultrasound contrast agent based on surfactants. The novel perfluoropropane-containing microbubbles were fabricated using ultrasonication of a sorbitan monostearate (Span 60) and polyoxyethylene 40 stearate (PEG40S). The obtained microbubbles distributed uniformly with an average diameter of 2.08±1.27μm. More than 99% of the microbubbles had a mean diameter less than 8μm, indicating that they were appropriately sized for intravenous administration as ultrasound contrast agent. The preliminary in vitro and in vivo ultrasound imaging study showed that such novel microbubbles demonstrated excellent enhancement under grey-scale pulse inversion harmonic imaging and power Doppler imaging. There were no abnormal changes of the rabbit’s basic life signs during the study. Based on the facts that we have fabricated micro-scale microbubbles, a nano-sized population with controlled mean diameter could be sorted by using centrifugal classifier from the parent polydisperse suspensions. Preliminary study with the nano-scale bubbles in vivo showed that such nanobubbles provided an excellent power Doppler enhancement. In order to understand the structure and stabilization mechanism of the novel microbubbles, Langmuir monolayer technique was performed to investigate the monolayer behaviour of microbubble forming components at molecular level. The results indicated that Span 60 could form a low surface tension monolayer which could diminish the gas diffusion from the core into the aqueous medium. The incorporation of PEG40S into the monolayer shell created a sub-energy barrier to prevent aggregation among microbubbles which impart good stability to the microbubbles. Based on the comparison ofπ-A isotherms for microbubbles and those obtained for mixtures of surfactants at different molar ratios, we suggested a composition in the microbubble skin close to nine molecules of Span 60 to each molecule of PEG40S. Polymeric microbubble ultrasound contrast agent (QDs@PLA microbubbles) with ultrasound/fluorescence bi-mode imaging functions was fabricated using double emulsion solvent evaporation and lyophilization methods. Polylactide (PLA), a biocompatible, safety and biodegradable high molecular polymer, was employed as the shell material and quantum dots (QDs) were employed as the luminescent material. The morphology of the polymeric microbubbles observed with scanning electron microscope appeared regular spherical with hollow structure and have a tight size distribution, which could meet the requirement of size for ultrasound contrast agent. The results of fluorescence emission spectrum and fluorescence microscope indicated that QDs@PLA microbubbles could maintain the good luminescence properties of QDs and PLA microbubble shell impart good fluorescence stability to the QDs. In vitro and in vivo ultrasound contrast effects indicated that QDs@PLA microbubbles showed high echogenicity. The fluorescence imaging in vitro and in vivo showed that QDs@PLA microbubbles exhibited excellent fluorescence imaging ability, and were in synchronism with ultrasound contrast effect.
引文
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