Ultra-high-frequency radio-frequency acoustic molecular imaging with saline nanodroplets in living subjects

Abstract

Molecular imaging is a crucial technique in clinical diagnostics but it relies on radioactive tracers or strong magnetic fields that are unsuitable for many patients, particularly infants and pregnant women. Ultra-high-frequency radio-frequency acoustic (UHF-RF-acoustic) imaging using non-ionizing RF pulses allows deep-tissue imaging with sub-millimetre spatial resolution. However, lack of biocompatible and targetable contrast agents has prevented the successful in vivo application of UHF-RF-acoustic imaging. Here we report our development of targetable nanodroplets for UHF-RF-acoustic molecular imaging of cancers. We synthesize all-liquid nanodroplets containing hypertonic saline that are stable for at least 2 weeks and can produce high-intensity UHF-RF-acoustic signals. Compared with concentration-matched iron oxide nanoparticles, our nanodroplets produce at least 1,600 times higher UHF-RF-acoustic signals at the same imaging depth. We demonstrate in vivo imaging using the targeted nanodroplets in a prostate cancer xenograft mouse model expressing gastrin release protein receptor (GRPR), and show that targeting specificity is increased by more than 2-fold compared with untargeted nanodroplets or prostate cancer cells not expressing this receptor.

Figure 1|. Experimental concept.

a, The spectral range of electromagnetic waves in the radio frequency range, where ultra-high frequency (UHF) ranges from 300 MHz to 1 GHz. b, Perspective view of the experimental setup for UHF-radio frequency acoustic (UHF-RF-acoustic) imaging of mice. The acoustic transducer was mounted on the side to image the x-y plane. The mouse was mounted vertically along the z axis. A translational stage can move the mouse in the +/−z direction and the rotational stage rotates the mouse in the x-y plane. Two RF antennae emit RF pulses (160 ns) to excite the UHF-RF-acoustic signals. The same setup was also used for scanning tissue phantoms. c, The side view (x-z plane) of the experimental setup. d, The top view (x-y plane) of the experimental setup. e, Point spread function of the UHF-RF-acoustic system, the enlarged view shows the UHF-RF-acoustic signal pattern generated with a point source. f, One-dimensional point spread function of the UHF-RF-acoustic system. The inset shows the full-width-at-half-maximum (FWHM) is 1.2 mm.

Figure 2|. Preparation of UHF-RF-acoustic contrast agents with engineered saline nanodroplets.

a, Experimental setup of the tube phantom for in vitro imaging with each tube containing different concentrations of electrolytes. b, UHF-RF-acoustic images of various types of electrolytes, the tubes contain 10 wt% concentration of NaCl, NaI, KCl, MgCl₂, and CaCl₂, and 4 wt% concentration of NaOH, with a diameter of 1 mm. c, UHF-RF-acoustic signal amplitude as a function of conductivity of the abovementioned electrolytes. The generated UHF-RF-acoustic peak amplitude follows a linear trend with the conductivity (grey dotted lines, R²=0.92). Data are presented as mean values +/− SD (N=5). d, A schematic illustration of nanodroplet synthesis using the double emulsion approach. e, Cryo-electron microscopy images show nanodroplets with NaCl_(aq) (25 wt%) core and perfluorocarbon shell (left) and a control droplet with only perfluorocarbon (right). f, Measured size distribution of the nanodroplets using dynamic light scattering, showing an average diameter of the nanodroplets ~ 250 nm. g, Stability test of nanodroplets with a median diameter of 250 nm with various shells, including △: soybean oil (control), □: perfluoropentane, ○: perfluorohexane, ◇: perfluoro-15-crown-5-ether, and ▽: perfluorodecalin. Data are presented as mean values +/− SD (N=25). By day 6, the soybean oil nanodroplets increase size by ~215% relative to day 0 (p < 0.0001). By day 14, the diameters of nanodroplets made of perfluorodecalin, perfluoro-15-crown-5-ether, perfluorohexane, and perfluoropentane nanodroplets increased by 18±15% (p = 0.2273), 28±17% (p = 0.0751), 65±17% (p < 0.0001), and 103±18% (p < 0.0001). h, Stability test of nanodroplets with perfluoropentane shell with different average diameters (□: 250 nm, : 450 nm, and ■: 800 nm). By day 14, 250 nm to 800 nm nanodroplets increases by 103±18% (p < 0.0001), 139±18% (p < 0.0001), 142±20% (p < 0.0001). Data are presented as mean values +/− SD (N=25).

Figure 3|. In vitro contrast-enhanced UHF-RF-acoustic imaging.

a, UHF-RF-acoustic signal amplitude of saline nanodroplets (open red circles) versus the initial concentration of NaCl_(aq), and the corresponding conductivity (filled blue squares, conductivity data adapted from Ref 27). Data are presented as mean values +/− SD (N=5). b, UHF-RF-acoustic signal amplitude versus nanodroplet concentration with encapsulated 25 wt% saline, showing a linear correlation (R²=0.995). Data are presented as mean values +/− SD (N=5). The grey dashed line shows the UHF-RF-acoustic signal level of physiological saline (0.9 wt%). c, UHF-RF-acoustic signal amplitude versus aging time over 14 days. Nanodroplets (1.8x10⁹ nanodroplets/mL) containing 25 wt% saline were measured, data are presented as mean values +/− SD (N=5). d, UHF-RF-acoustic signal amplitude of tube phantoms with a 1-mm diameter. Six tubes were imaged; four tubes contain nanodroplets (1×10⁹ nanodroplets/mL); one contains 20-nm gold nanoparticles (AuNPs, 5×10¹¹ nanoparticles/mL); and one contains 100-nm iron oxide nanoparticles (Fe₃O₄, 5×10¹¹ nanoparticles/mL). Both AuNPs and Fe₃O₄ were prepared at 500× higher concentration than the nanodroplets to bring their UHF-RF-acoustic signals above the noise level. The 1× nanodroplets produce 3.2 ± 0.7 times higher UHF-RF-acoustic signals than the 500× Fe₃O₄ nanoparticles (*, p=0.0002, N=5), and 5.1± 0.6 times higher signals than the 500× gold nanoparticles (**, p=1.4×10⁻⁷, N=5). Data are presented as mean values +/− SD. e, UHF-RF-acoustic signal amplitude of nanodroplets as a function of imaging depth in bovine tissue (photograph with setup on the left). Six 3-mm-diameter inclusions were imaged, located at 0.5 cm, 1.8 cm, 2 cm, 2.8 cm, 3.5 cm, and 5 cm from the tissue surface. Each inclusion contains 1:1 volume ratio of nanodroplets (3×10⁹ nanoparticles/mL with 25 wt% saline) and 12% gelatin. Data are presented as mean values +/− SD (N=5). f, UHF-RF-acoustic imaging of a Stanford logo phantom. The phantom contains nanodroplets (8×10⁹ nanodroplets/mL); the inclusion rotates for tomographic imaging.

Figure 4|. GRPR-targeted nanodroplets showed specific targeting to prostate cancer cells and low toxicity in cell culture.

a, Extinction spectrum of the nanodroplets shows the optical absorption peak from indocyanine green (ICG) dye. b, Western blot analysis for GRPR expression in different prostate cancer cell lines. c, Cell viability test using non-targeted nanodroplets, showing no obvious reduction in cell viability when cells were incubated with up to 1×10¹¹ nanodroplets/mL (p=0.958 relative to control), and low cell toxicity up to 3×10¹¹ nanodroplets/mL (*, p=0.009, N=30) at 24 hours post incubation relative to cells incubated in the absence of nanodroplets. Data are presented as mean values +/− standard deviation (N=30). d, Optical fluorescence imaging of ICG-labeled targeted (column 1) and non-targeted (column 2) nanodroplets incubated with GRPR⁺ (PC3) and GRPR⁻ (DU145) cells at 12 hours post incubation. Column 3 shows the optical fluorescence imaging of ICG-labeled targeted nanodroplets incubated with antibody-pre-blocked PC3 and DU145 cells, also at 12 hours post incubation. Both PC3 and DU145 cells express green fluorescent protein (GFP). ICG dyes on nanodroplets and GFP of the cells were shown as red and green, respectively.

Figure 5|. In vivo UHF-RF-acoustic molecular imaging using targeted nanodroplets.

a, Configuration of the tomographic mouse imaging. b, In vivo UHF-RF-acoustic imaging with subcutaneous GRPR⁺ tumors (PC3) using targeted nanodroplets (group 1, N=5), GRPR⁻ tumors (DU145) using targeted nanodroplets (group 2, N=5), PC3 tumors using non-targeted nanodroplets (group 3, N=5), and DU145 tumors using non-targeted nanodroplets (group 4, N=5). c, Comparison of UHF-RF-acoustic signal amplitude in the tumor region with targeted and non-targeted nanodroplets, showing the strongest signals from the targeted nanodroplets with PC3 tumors (*, p=0.007; **, p=0.404). Data are presented as mean values +/− SD (N=5). d, Epifluorescence imaging of mice in groups 1 to 4 at 48 hours post injection, showing the strongest ICG fluorescence signals from PC3 tumor bearing mouse with the GRPR-targeted nanodroplets. The scanning area is 66 mm × 50 mm. e, Fluorescence imaging (top) of harvested main organs and tumor from one mouse in group 1, showing that the nanodroplets mainly accumulate at the tumor and the spleen. Fluorescence imaging (bottom) of the harvested tumors from one mouse from each group, showing the strongest signal from the mouse in group 1, demonstrating their highest targeting specificity and efficiency. f, ICG fluorescence signals from the major organs and tumors from the 4 groups of mice, indicating that the nanodroplets mainly accumulate at the tumor and the spleen, but only GRPR-targeted nanodroplets show highest binding specificity at the tumor site (*, p=0.006; **, p=0.0005; ***, p=0.0007; ****, p=0.017; and *****, p=0.051). Data are presented as mean values +/− SD (N=5). g, Immunohistochemical tissue sections of liver, kidney, and spleen from mice with non-targeted nanodroplet or saline injections (100 μL, 1×10¹¹ nanodroplets/mL) for 2 weeks, stained with hematoxylin and eosin (H&E) and Perls’ Prussian Blue. There was no noticeable tissue damage from the intravenous nanodroplet injections.