Multiplex Ultrasound Imaging of Perfluorocarbon Nanodroplets Enabled by Decomposition of Postvaporization Dynamics.

Despite the real-time, nonionizing, and cost-effective nature of ultrasound imaging, there is a dearth of methods to visualize two or more populations of contrast agents simultaneously─a technique known as multiplex imaging. Here, we present a new approach to multiplex ultrasound imaging using perfluorocarbon (PFC) nanodroplets. The nanodroplets, which undergo a liquid-to-gas phase transition in response to an acoustic trigger, act as activatable contrast agents. This work characterized the dynamic responses of two PFC nanodroplets with boiling points of 28 and 56 °C. These characteristic responses were then used to demonstrate that the relative concentrations of the two populations of PFC nanodroplets could be accurately measured in the same imaging volume within an average error of 1.1%. Overall, the findings indicate the potential of this approach for multiplex ultrasound imaging, allowing for the simultaneous visualization of multiple molecular targets simultaneously.

Dual-drug loaded ultrasound-responsive nanodroplets for on-demand combination chemotherapy.

Phase-changing nanodroplets are nanometric sized constructs that can be vaporized via external stimuli, such as focused ultrasound, to generate gaseous bubbles that are visible in ultrasound. Their activation can also be leveraged to release their payload, creating a method for ultrasound-modulated localized drug delivery. Here, we develop a perfluoropentane core nanodroplet that can simultaneously load paclitaxel and doxorubicin, and release them in response to an acoustic trigger. A double emulsion method is used to incorporate the two drugs with different physio-chemical properties, which allows for a combinatorial chemotherapy regimen to be used. Their loading, release, and biological effects on a triple negative breast cancer mouse model are investigated. We show that activation enhances the drug-delivery effect and delays the tumor growth rate in vivo. Overall, the phase-changing nanodroplets are a useful platform to allow on-demand delivery of combinations of drugs.

Fluorescent Phase-Changing Perfluorocarbon Nanodroplets as Activatable Near-Infrared Probes.

The sensitivity of fluorescence imaging is limited by the high optical scattering of tissue. One approach to improve sensitivity to small signals is to use a contrast agent with a signal that can be externally modulated. In this work, we present a new phase-changing perfluorocarbon nanodroplet contrast agent loaded with DiR dye. The nanodroplets undergo a liquid-to-gas phase transition when exposed to an externally applied laser pulse. This results in the unquenching of the encapsulated dye, thus increasing the fluorescent signal, a phenomenon that can be characterized by an ON/OFF ratio between the fluorescence of activated and nonactivated nanodroplets, respectively. We investigate and optimize the quenching/unquenching of DiR upon nanodroplets' vaporization in suspension, tissue-mimicking phantoms and a subcutaneous injection mouse model. We also demonstrate that the vaporized nanodroplets produce ultrasound contrast, enabling multimodal imaging. This work shows that these nanodroplets could be applied to imaging applications where high sensitivity is required.

EGFR-Targeted Perfluorohexane Nanodroplets for Molecular Ultrasound Imaging.

Perfluorocarbon nanodroplets offer an alternative to gaseous microbubbles as contrast agents for ultrasound imaging. They can be acoustically activated to induce a liquid-to-gas phase transition and provide contrast in ultrasound images. In this study, we demonstrate a new strategy to synthesize antibody-conjugated perfluorohexane nanodroplet (PFHnD-Ab) ultrasound contrast agents that target cells overexpressing the epidermal growth factor receptor (EGFR). The perfluorohexane nanodroplets (PFHnD) containing a lipophilic DiD fluorescent dye were synthesized using a phospholipid shell. Antibodies were conjugated to the surface through a hydrazide-aldehyde reaction. Cellular binding was confirmed using fluorescence microscopy; the DiD fluorescence signal of the PFHnD-Ab was 5.63× and 6× greater than the fluorescence signal in the case of non-targeted PFHnDs and the EGFR blocking control, respectively. Cells were imaged in tissue-mimicking phantoms using a custom ultrasound imaging setup consisting of a high-intensity focused ultrasound transducer and linear array imaging transducer. Cells with conjugated PFHnD-Abs exhibited a significantly higher (p < 0.001) increase in ultrasound amplitude compared to cells with non-targeted PFHnDs and cells exposed to free antibody before the addition of PFHnD-Abs. The developed nanodroplets show potential to augment the use of ultrasound in molecular imaging cancer diagnostics.

Repeated Acoustic Vaporization of Perfluorohexane Nanodroplets for Contrast-Enhanced Ultrasound Imaging.

Superheated perfluorocarbon nanodroplets are emerging ultrasound imaging contrast agents that boast biocompatible components, unique phase-change dynamics, and therapeutic loading capabilities. Upon exposure to a sufficiently high-intensity pulse of acoustic energy, the nanodroplet's perfluorocarbon core undergoes a liquid-to-gas phase change and becomes an echogenic microbubble, providing ultrasound contrast. The controllable activation leads to high-contrast images, while the small size of the nanodroplets promotes longer circulation times and better in vivo stability. One drawback, however, is that the nanodroplets can only be vaporized a single time, limiting their versatility. Recently, we and others have addressed this issue by using a perfluorohexane core, which has a boiling point above body temperature. Thus after vaporization, the microbubbles recondense back into their stable nanodroplet form. Previous work with perfluorohexane nanodroplets relied on optical activation via pulsed laser absorption of an encapsulated dye. This strategy limits the imaging depth and temporal resolution of the method. In this study, we overcome these limitations by demonstrating acoustic droplet vaporization with 1.1-MHz high-intensity focused ultrasound (HIFU). A short-duration, high-amplitude pulse of focused ultrasound provides a sufficiently strong peak negative pressure to initiate vaporization. A custom imaging sequence was developed to enable the synchronization of a HIFU transducer and a linear array imaging transducer. We show a visualization of repeated acoustic activation of perfluorohexane nanodroplets in polyacrylamide tissue-mimicking phantoms. We further demonstrate the detection of hundreds of vaporization events from individual nanodroplets with activation thresholds well below the tissue cavitation limit. Overall, this approach has the potential to result in reliable and repeatable contrast-enhanced ultrasound imaging at clinically relevant depths.