Ultrasound signal from sub-micron lipid-coated bubbles

Nanoscale ultrasound agents have recently gained interest in cancer imaging and therapy for their ability to exploit the enhanced permeability and retention (EPR) effect in tumors. There have been numerous reports of echogenic nanoscale bubbles, but it remains unclear whether these sub-micron bubbles are responsible for producing the observed acoustic backscatter at clinically relevant frequencies. Because nanobubble polydispersity is high, larger microbubbles are thought to be the significant contributors to the observed signal. Accordingly, this study examined echogenicity of lipid- and surfactant-stabilized perfluoropropane nanobubbles following extra steps to ensure larger bubbles were removed from solution. Microbubbles were separated from nanobubbles based on their buoyancy by centrifugation at 50g for 5 min. Isolated nanobubbles were also filtered using track-etched polycarbonate (PCTE) filters with a pore size of 1.0 micron, 0.6 micron, and 0.4 micron using a syringe pump at 100 microliters/min and were imaged in a custom agarose mold in PBS using contrast harmonic imaging (Toshiba, 12 MHz, MI 0.1). Nanobubbles isolated via centrifugation were found to have considerable echogenicity at 12 MHz. Resonant mass measurement results indicate that these bubbles were below 1 micron in diameter and typical size range was between 100 to 600 nm (mean diameter 233 ± 5 nm). A reduction in signal after filtration was noted, but NB activity was observed under all conditions. This study demonstrates that sub-micron bubbles are capable of producing strong echogenic signals at clinically used ultrasound frequencies.