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.
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.
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