Lipid-stabilized monodisperse microbubbles produced by flow focusing for use as ultrasound contrast agents and targeted drug delivery.

摘要

Lipid-encapsulated microbubbles have demonstrated utility in biomedical applications as ultrasound contrast agents and drug delivery vehicles. Current production methods of these microbubbles result in distributions with a large size variance. The size and monodispersity of ultrasound contrast agents are fundamentally important due to the relationship between bubble diameter and resonant frequency, destruction threshold, and susceptibility to radiation force. Several groups have recently developed microfluidic technologies for generation of microbubbles, but to date no group has demonstrated the production of shell-stabilized monodisperse contrast agents using these techniques. In this research, we use microfluidic-based flow focusing methods to produce monodisperse microbubbles in the diameter range required for in-vivo imaging. The diameter of the bubbles produced using this technique can be precisely tailored by adjusting the gas and liquid flow rate parameters. In order to stabilize the microbubbles for use as ultrasound contrast agents, we examine aqueous mixtures of glycerol/propylene glycol as well as stabilizing lipids. Fluorescence microscopy, high-speed camera imaging, and particle size analysis were used to investigate the roles of lipid phase behavior, filling and surrounding gas dissolution, Ostwald ripening, and coalescence in the stability of microbubbles formed by flow focusing. Monodisperse microbubbles coated with a lipid-shell in a viscous solution were found to be stable up to three months.; Generally, contrast agents are injected intravascularly, and are retained on the endothelium at the site of pathology via adhesion ligands incorporated into the agent shell. The production of a functionalized, lipid-encapsulated, microbubble contrast agent with a monodisperse population is demonstrated, and we evaluate parameters, which influence the size distribution and demonstrate initial acoustic testing. We have demonstrated that these contrast agents can be functionalized to present biotin ligands in the shell to which antibodies or other targeting ligands can be secured through an avidin linker. We also investigate the sensitivity of polydisperse microbubbles to hydrostatic pressure during the injection process, by injecting microbubbles into tubing using various rates, catheter sizes, and microbubble concentrations. Our results indicate that the concentration and size distribution of microbubbles can be substantially altered in cases of rapid injection through small needles.