Phase Behavior of Mixed Lipid Monolayers on Perfluorocarbon Nanoemulsions and its Effect on Acoustic Contrast

Lipid-stabilized nanoemulsions containing a volatile liquid perfluorocarbon core have been studied as ultrasound contrast agents owing to their ability to transform into high-contrast microbubbles when subjected to high intensity focused ultrasound (HIFU). However, while there have been several studies on the effect of acoustic parameters on contrast, the effect of the droplet's stabilizing shell has not been studied as extensively. Inspired by previous studies showing lateral phase separation in microbubbles and vesicles, nanodroplets were formulated with a perfluorohexane core and a shell composed of varying amounts of saturated (DPPC) phospholipids, unsaturated (DOPC) phospholipids, and cholesterol, which were fractionated to obtain nanodroplets of mean diameter 300-400 nm and were stable over one week. When the DOPC content was increased to 40 mol%, ultrasound contrast increased by about one order of magnitude over DPPC-only droplets. Based on fluorescence microscopy results of lateral lipid phase separation on the droplet surface, the various combinations of DPPC, DOPC, and cholesterol were assigned to three regimes on the ternary phase diagram: solid-liquid ordered (low contrast), liquid ordered-liquid disordered (medium contrast), and solid-liquid disordered (high contrast). These regimes were confirmed by TEM analysis of nanoscale droplets. Droplets containing mixed lipid monolayers were also found to produce a significantly greater yield than single-component droplets. The discovery of the dependence of acoustic response on lipid phase separation will help to understand the formulation, behavior, and vaporization mechanism of acoustically-responsive nanoemulsions.