Lyophilized perfluorocarbon nanodroplets: toward scalable and stable acoustic contrast agents for ultrasound biomedical applications

Perfluorocarbon nanodroplets are emerging ultrasound-responsive agents with strong potential for diagnostic imaging and therapeutic applications due to their ability to undergo acoustic droplet vaporization. However, their inherent physicochemical instability limits their long-term storage and clinical translation. Here, we report a scalable freeze-drying protocol for lipid-stabilized perfluorocarbon nanodroplets to ensure long-term stability without compromising NDs characteristics and acoustic performance. In this study, nanodroplets composed of perfluorobutane or perfluoropentane were produced by microfluidic nanoprecipitation and protected by cryoprotectants. The influence of cryoprotectant type on cake appearance, particle size, polydispersity index, chemical composition and vaporization behavior was systematically evaluated. Chemical integrity was assessed by gas chromatography for perfluorocarbon core and ethanol content and by Karl Fischer titration for residual water. Acoustic performance was determined by measuring ADV thresholds under focused ultrasound. Disaccharide-based cryoprotectants, particularly trehalose at 10 wt%, provided optimal protection, preserving nanodroplet size (200-250 nm), monodispersity (Polydispersity Index <0.2), and acoustic responsiveness after freeze-drying. In addition, quantitative analyses showed that the chemical composition of nanodroplets was maintained, with perfluorocarbon and ethanol contents remaining unchanged, while residual water content stayed below 2 %, indicating effective preservation of the freeze-dried product. Stability studies demonstrated preservation of particles, chemical and acoustic properties for at least six months at 4 °C and 25 °C. These results establish a robust strategy for stabilizing perfluorocarbon nanodroplets and support their practical deployment as ultrasound-responsive agents for biomedical applications.