The Effect of Freeze/Thawing on the Physical Properties and Acoustic Performance of Perfluoropropane Nanobubble Suspensions

Nanobubble (NBs) contrast agents have the potential to augment the molecular imaging capabilities of ultrasound (US), especially in cancer imaging. However, as with most bubble-based formulations, these shell-stabilized perfluoropropane (C3F8) bubbles are fragile, last only several hours after self-assembly, and size isolation. Longer storage of the NB preparation leads to bubble dissolution. The short shelf-life after activation has limited broader bubble adoption and can hamper the diagnostic and therapeutic potential. Distribution of the NBs is also limited by the access to the amalgamator and centrifuge used for NB activation and isolation. In this work, we tested two simple freeze/thaw strategies for long term NB storage and examined their effect on NB size and concentration, initial acoustic activity, and US signal decay. Aliquoted NBs were either stored at -80°C for gradual cooling (GC-80), or flash frozen by submerging in liquid nitrogen prior storing at -80°C (FF-80). Our results suggest that NB suspension could be stored at -80°C for at least 28 days with only minor change in physical properties and acoustic performance. FF-80 NBs exhibited preserved NB size distribution and acoustic activity for over 28 days. GC-80 NBs showed a narrower size distribution but decrease in NB concentration at day 28. These differences are likely a result of shell composition changes induced by the freezing process, leading to less stable bubbles. The specific mechanism for the observed changes will be evaluated in future studies.