Comparison of ultrasound contrast agent production by sonication and mechanical agitation

Microbubbles (MBs) are surfactant-coated gas vesicles used widely in ultrasound imaging and increasingly for therapy. MBs can be produced by a range of methods, most commonly involving sonication and/or agitation. This study aimed to compare MB production by agitation with a bead mill tissue homogeniser or with a dental amalgamator, and sonication. All methods produced MBs with a similar size distribution (10-90% inter-percentile range ∼0.44-1.6 μm), but both the MBs produced by the agitation methods had higher concentration than those produced by sonication (∼10¹⁰ vs ∼10⁹ particles/mL), and a more persistent ultrasound response. The lipid order of the phospholipid coating was investigated spectroscopically, and it was found that MBs produced by all three methods had a similar generalised polarisation. The effect of varying agitation frequency and duration on MB size distribution and concentration was investigated, with increases in either parameter raising MB concentration and reducing mean size, each showing distinct limiting behaviour. Lastly, the feasibility of generating larger MB volumes with the tissue homogeniser was investigated, yielding MBs of comparable size and concentration in both 2 mL and 5 mL vials. The results of this study suggest that bead mill homogenisers can generate MBs with comparable size distribution, stability and shell lipid order to those generated using dental amalgamators. At low lipid concentration (0.9 mg/mL), both methods outperform sonication; with the added advantage that there is a lower risk of contamination. The tissue homogeniser offers further advantages compared with dental amalgamators, in terms of MB production volume and reliability.