Beyond a Single ADV Threshold: Toward Reproducible Production and Comprehensive Acoustic Characterization of...

Perfluorocarbon (PFC) nanodroplets are emerging as promising ultrasound-responsive phase-change contrast agents that enable acoustically triggered vaporization for imaging and drug delivery. However, reported acoustic droplet vaporization (ADV) thresholds vary widely across studies, reflecting differences in formulation, experimental setups, and threshold definitions, which complicates quantitative comparison and rational nanodroplet design. This work investigates how process and formulation parameters influence nanodroplet size, polydispersity, and vaporization behavior, with the aim of improving the consistency, comparability, and interpretability of ADV measurements.

Lipid-shelled PFC nanodroplets were produced using a commercially available microfluidic platform, enabling controlled tuning of droplet size, polydispersity, as well as core and shell composition. Vaporization behavior was evaluated across a range of environmental and formulation parameters, including surrounding medium temperature, perfluorocarbon core, lipid saturation, and phosphatidylethanolamine (PE)-PEG content. The resulting vaporization curves were analyzed using four commonly applied ADV threshold definitions: two onset-based and two population-based metrics.

Microfluidics enables highly reproducible production of PFC nanodroplets with narrow size distributions, even for highly volatile perfluorobutane cores. ADV thresholds and corresponding ultrasound signal intensities depend strongly on both the shape of the vaporization curve and the chosen analysis metric. Onset-based definitions capture low-pressure, sparse vaporization events, whereas population-based metrics reflect the overall population response and more reliably reveal formulation-dependent effects, including subtle modifications to the shell architecture.

While traditional single-value onset metrics effectively capture early, sparse vaporization events, they may not fully predict in vivo performance, which often relies on bulk population activation. Instead, we suggest reporting full vaporization curves and their corresponding fitting parameters to ensure a more comprehensive acoustic characterization. This integrated framework unites reproducible microfluidic PFC nanodroplet production with standardized evaluation, helping to bridge the gap toward clinical translation.