The Effect of Nanobubble Ultrasound Contrast Agent Shell Stiffness and Temperature on Stability and Interactions with Red Blood Cells

Lipid-shell C3F8 nanobubble (NB) ultrasound contrast agents have demonstrated an extended in vivo lifespan compared to traditional microbubble contrast agents. One potential explanation behind this extended lifespan is non-covalent interactions between NBs and red blood cells (RBCs). This has been observed in vitro where interactions increased contrast intensity and signal stability over time. However, the mechanism of this interaction and the factors influencing it have not been fully elucidated. In this study, we investigate the role of NB shell stiffness and solution temperature on signal stability in human whole blood and PBS in vitro. We used three NB shell stiffnesses and a fourth formulation with the addition of a targeting ligand and assessed extent of RBC interaction using autocorrelation analysis. Results demonstrate a clear dependence of RBC interaction on shell stiffness. The stiffest NB formulation had a decorrelation time ~2x faster than all other formulations and showed no effect on signal change over time. Less stiff NBs resulted in an increase in intensity over time and increased decorrelation time, consistent with prior studies. No such effects were observed in PBS. Furthermore, the presence of a targeting ligand did not appear to play a role in contrast enhancement over time or decorrelation time. Decorrelation time in whole blood decreased with increasing temperature. This work examined the effect of shell stiffness and solution temperature on NB signal stability and interactions with RBCs in human whole blood in vitro. Understanding why NBs interact with RBCs could improve NB formulation optimization for extended circulation time in vivo.