Time-dependent size stability and shell behaviors of lipid-coated microbubbles

Microbubbles are excellent contrast-enhancing agents for ultrasound imaging. A long shelf life with a robust size distribution is critical for their efficacy. Here, we investigated the long-term stability and attenuation of a custom-made polydisperse microbubble suspension. The microbubbles were prepared using mechanical agitation with a gas core of perfluorobutane (C4F10), and a 9:1 molar ratio mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-dipalmitoyl-sn-glycero3-phosphatidylethanolamine-polyethyleneglycol-2000 (DPPE-PEG2000) lipids. Their size distribution and attenuation response were measured in regular intervals over 30 days. The size remained the same (∼2.25 μm) for the first 13 days before slightly increasing to ∼2.5 μm. The microbubble concentration decreased with time (7.14 ± 1.12 × 109 MB/mL initially and 3.29 ± 0.66 × 109 MB/mL at day 30), resulting in a corresponding decrease in attenuation. We determined the shell properties of microbubbles by applying the exponential elasticity model (EEM) to the attenuation. Like the size, the shell elasticity and viscosity remained unchanged for 13 days and then increased by ∼50% and ∼200%, respectively. The study sheds light on the shelf life and in vitro stability of lipid-coated microbubbles, offering valuable information about their effectiveness as ultrasound contrast agents.