EGFR-Targeted Perfluorohexane Nanodroplets for Molecular Ultrasound Imaging

Abstract

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.

Keywords: acoustic droplet vaporization; molecular targeting; perfluorocarbon nanodroplet; ultrasound imaging.

Figure 1

The antibodies were first labeled with AF 555 antibody labeling kit. Second, the Fc region of the antibody was oxidized by reacting with 100-mM NaIO₄ leading to aldehydes on the Fc region. The aldehydes on the activated Fc region then bind to the hydrazides available on the surface of the PFHnDs, forming a stable hydrazone bond and leading to the final antibody conjugated nanodroplet (PFHnD-Ab). The core of the PFHnD contains perfluorohexane (b.p. 56 °C) and DiD fluorescent dye.

Figure 2

Schematic of the custom ultrasound setup. The single element HIFU transducer uses a polyacrylamide coupling cone to deliver the activation energy needed to induce the PFHnD-Ab phase change. Polyacrylamide phantoms containing PFHnD-Abs conjugated to cells were placed on the 3D printed stage. A linear array B-mode transducer was placed perpendicular to the activation plane to capture the vaporization and recondensation processes.

Figure 3

(A) The size distribution of both the non-targeted PFHnDs (534.2 ± 37.5 nm; n = 3) and antibody conjugated PFHnD-Ab (641 ± 48.5 nm; n = 3). (B) The zeta potential of both targeted (PFHnD-Ab; −8.88 ± 3.54 mV; n = 3) and non-targeted (PFHnD; −12.7 ± 2.01 mV; n = 3) nanodroplets.

Figure 4

(A) FaDu cells were imaged using a fluorescent microscope for the different groups; cells, cells with targeted PFHnD-Abs, cells with non-targeted PFHnDs, and cells that were exposed to unlabeled free antibody prior to incubating with targeted PFHnD-Abs (blocking study). DiD lipophilic dye was embedded into the PFHnD-Ab and PFHnDs, while the antibodies were fluorescently labeled with AF 555. Dark-field cell images were used to identify the location of each cell in the image. The locations were then used to compute the fluorescent signal within the cell. Merged images show the two fluorescent channels (DiD and AF 555) overlaid. (B) The mean DiD and AF 555 fluorescence per cell for each group was computed from the fluorescent images, resulting in the PFHnD-Ab group having a statistically significant (*** = p < 0.001) higher mean DiD and AF 555 fluorescence/cell compared to the all other groups. There was not any statistical significance between the control groups.

Figure 5

The PFHnD-Abs were subjected to either 0, 10, 100, or 1000 vaporization-recondensation cycles using HIFU before being incubated with cells to test the effects of activation on cellular binding. (A) Fluorescent images of cells after incubation with the pre-activated PFHnD-Abs. (B) The mean fluorescence per cell in the DiD channel showed no statistically significant difference except for two isolated cases (* p < 0.01) using the Mann-Whitney Test. (C) The mean fluorescence per cell in the AF 555 channel resulted in isolated cases of statistically significant differences using the Mann-Whitney test (* p < 0.01). (D) Before and after images of cells with PFHnD-Abs bound to the surface before HIFU activation in the DiD and AF 555 channels. (E,F) The mean fluorescence per cell before and after HIFU activation did not show any statistical difference in the DiD and AF 555 channel respectively.

Figure 6

Ultrasound images of cells embedded in polyacrylamide tissue-mimicking phantoms before and after the HIFU activation of the PFHnDs. (A) The difference between the two images is displayed in the right panel, indicating PFHnD activation. Cells conjugated with the targeted droplets (PFHnD-Ab) were imaged along with the control groups; non-targeted droplets (PFHnD), blocking group (Free antibody and PFHnD-Ab), and FaDu cells without PFHnDs. (B) The differential amplitude was calculated for each of the groups and resulted in the PFHnD-Ab group having a significantly higher differential amplitude (*** p < 0.001) than the other groups. (C) The average ultrasound amplitude within the focal spot for each ultrasound frame captured for each of the groups. The HIFU was pulsed prior to frames 7, 9, 11, 13, and 15, resulting in the ultrasound amplitude spike within those frames.

Figure 7

Cells with PFHnD-Abs attached to them were diluted to different concentrations and embedded into polyacrylamide phantoms. (A) Images of the differential ultrasound amplitude for each of the different concentrations. The expected cells/focal area was calculated based on the initial concentration of cells. (B) The differential amplitude plotted as a function of the expected cells/focal area yielded an r² of 0.998.