Fabrication, Properties And Applications Of Novel Microbubble Ultrasound Contrast Agents

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.