Novel applications for contrast ultrasound : the development of microbubble agents for site-targeted imaging and gene delivery

Abstract

Contrast-enhanced ultrasound (CEU) is a rapidly emerging technique in the field of non-invasive cardiac imaging. CEU is performed by ultrasound imaging of gas-filled encapsulated microbubbles that cause backscatter of ultrasound during their transit through the microcirculation. The potential uses for contrast-enhanced ultrasound are expanding beyond blood pool enhancement and perfusion imaging. This thesis investigates the two most promising areas of future application: site-targeted imaging and therapeutic gene delivery. Novel target-specific microbubbles were developed and their microvascular behaviour assessed. In vivo site-targeted non-invasive imaging using these unique microbubbles was demonstrated in three different settings. Ultrasound imaging of microbubbles targeted to activated leukocytes accurately assessed the severity and spatial extent of myocardial inflammation following ischaemia-reperfusion injury. The degree of endothelial P-selectin expression following renal ischaemia-reperfusion injury was assessed using ultrasound imaging of microbubbles targeted to P-selectin. The extent of tissue angiogenesis, as represented by the endothelial expression of the integrin receptor αvβ3 in neovessels, was assessed using αv-targeted microbubbles. Imaging of angiogenesis was demonstrated in both a growth factor driven and an ischaemic model. Contrast enhanced ultrasound shows significant promise as a means to non-invasively assess the endothelial expression of adhesion molecules in a range of disease states. The abrupt fragmentation of microbubbles using ultrasound facilitates the extravascular sojourn of genetic material and pharmacologic agents. Novel cationic microbubbles were conjugated with plasmid DNA, and in vivo ultrasound destruction of these microbubbles in the microvasculature lead to tissue-specific and reliable transfection of myocardial and skeletal muscle tissue. Transfection was more effective following intra-arterial administration of microbubbles. Microbubble destruction resulted in substantial extravascular plasmid deposition without significant microvessel injury, suggesting microporation as the probable mechanism of plasmid delivery. Contrast enhanced ultrasound has considerable potential for application in both the research and clinical arenas as a non-invasive technique for molecular imaging, and as a means of achieving targeted delivery of genetic material to muscle tissue.