Microbubble composition and preparation for high-frequency contrast-enhanced ultrasound imaging: in vitro and in vivo evaluation

Although high-frequency ultrasound imaging is gaining attention in various applications, hardly any ultrasound contrast agents (UCAs) dedicated to such frequencies (>15 MHz) are available for contrast-enhanced ultrasound imaging (CEUS). Moreover, the composition of the limited commercially available UCAs for high-frequency CEUS (hfCEUS) is largely unknown, while shell properties have been shown to be an important factor for their performance. The aim of our study was to produce UCAs in-house for hfCEUS. Twelve different UCA formulations A-L were made by either sonication or mechanical agitation. The gas core consisted of C4F10 and the main coating lipid was either DSPC (A-F formulation) or DPPC (G-L formulation). Mechanical agitation resulted in UCAs with smaller microbubbles (number weighted mean diameter ~1 μm) than sonication (number weighted mean diameter ~2 μm). UCA formulations with similar size distributions but different main lipid component, showed that the DPPC-based UCA formulations had higher nonlinear responses at both the fundamental and subharmonic frequencies in vitro for hfCEUS using the Vevo2100 high-frequency preclinical scanner (FUJIFILM VisualSonics Inc.). In addition, UCA formulations F (DSPC-based) and L (DPPC-based) that were made by mechanical agitation performed similar in vitro to the commercially available Target-Ready MicroMarker (FUJIFILM VisualSonics Inc.). UCA formulation F also performed similar to Target-Ready MicroMarker in vivo in pigs with similar mean contrast intensity within the kidney (n=7), but formulation L did not. This is likely due to the lower stability of formulation L in vivo. Our study shows that DSPC-based microbubbles produced by mechanical agitation resulted in small microbubbles with high nonlinear responses suitable for hfCEUS imaging.