Ultrasound-mediated microbubble-membrane interaction

Abstract

In near future, personalized gene therapy and cancer immunotherapy is a promising, emerging method of medical treatment. Drug delivery mechanism employing ultrasound to induce cell membrane disruption, termed sonoporation, is an attractive solution for the aforementioned application due to the non-invasive nature. Microbubbles which are conventionally used for enhancing ultrasound imaging contrast, found new purposes in the drug delivery application, i.e. as cavitation nuclei at targeted delivery site and drug carrier via the microbubble shells. The thesis primarily studies the interaction between cell membrane and microbubble, which is necessary to improve the efficiency of microbubble-assisted sonoporation-based drug delivery.

Experimental methodologies and protocols involved in completing the proposed research include cell cultivation and preparation, artificial cell membrane model preparation by electroformation, targeting microbubble preparation and cavitation phase detection, ultrasound exposure field calibration, ultrasound exposure apparatus set-up, confocal imaging protocols and image analysis, and western blot protein expression assay.

As an attempt to gain new insight for developing and utilizing microbubble for ultrasound-mediated drug delivery, this thesis first explores the real-time dynamics of microbubble-membrane interaction on live ZR-75-30 breast cancer cells upon ultrasound implementation. A single site sonoporation protocol is adopted at single-cell level, allowing real-time monitoring of the membrane resealing dynamics following sonoporation. In this study, membrane blebbing is demonstrated to be a repair maneuver for injured plasma membrane, and microbubble diameter was found correlated to sonoporated pore size as well as primary bleb volume that appear at recovering sonoporated site. Next, similar experiments are performed on giant lipid vesicle, which is an artificial cell membrane model to study whether membrane forces alone, without the biological factors, can lead to membrane recovery after a sonoporation event. Influx dynamics of fluorescent tracer pre-loaded in extracellular buffer into the giant vesicle during a sonoporation event is also studied. It was found that larger microbubble diameter would induce larger pore size on giant vesicle membrane, as well as higher transmembranous diffusion rate via the sonoporated pore. Besides, presence of cholesterol was found to be essential in facilitating membrane recovery, presumably due to specific interactions between the phospholipid and cholesterol molecules, which subsequently alters the membrane physical properties. Finally Western blot assay is carried out to quantify the changes of six stress-related protein expressions in sonoporated cells at macroscale level instead of single-cell level. The final findings indicate that sonoporation has elicited a downstream stress response in sonoporated cells, lasting up to 24 hours post-sonoporation.