In Vitro and In Vivo Behavioral Evaluation of Condensed Lipid-Coated Perfluorocarbon Nanodroplets

Abstract

BackgroundPhase-shift contrast agents consist of a liquid perfluorocarbon core that can be vaporized by ultrasound to generate echogenic contrast with excellent spatiotemporal control. The purpose of the present work was to evaluate the in vitro and in vivo behavior of condensed lipid-shelled nanodroplets using different analytical procedures. MethodsIn this study, perfluorobutane nanodroplets were prepared by condensation of precursor fluorescently labeled lipid shelled microbubbles and were characterized in terms of size distribution, gas core content, in vitro stability in blood as well as for their acoustic vaporization behavior using a custom-made set-up. In particular, their in vivo behavior was thoroughly investigated after intravenous bolus injection in rats. To this end, we report, for the first time, the efficient use of three complementary detection procedures to assess the NDs in vivo persistence namely 1) Ultrasound contrast imaging of vaporized NDs, 2) Gas chromatography-mass spectrometry to determine the perfluorobutane core content and 3) Fluorescence intensity measurement in the collected blood samples. ResultsThe Coulter Counter Multisizer results confirmed the size distribution shift post-condensation. Furthermore, similar PFB concentration from MBs and NDs suspensions was obtained, thus demonstrating an exceptionally low rate of microbubbles breaking and spontaneous nanodroplets vaporization. As expected, these nanoscale droplets display longer circulation time compared to clinically approved microbubbles and only slight variations of half-life were observed between the three monitoring procedures. Finally, echogenic signal observed in focal area of the liver and spleen after vaporization was confirmed by accumulation of fluorescent nanodroplets in these organs. ConclusionThese results further contribute to deepen our understanding of both in vitro and in vivo behavior of sonoresponsive nanodroplets, which is key to enable efficient clinical translation.