Effects of shell-integrated Sudan Black dye on the acoustic activity and ultrasound imaging properties of lipid-shelled nanoscale ultrasound contrast agents

Abstract

Significance: An effective contrast agent for concurrent multimodal photoacoustic (PA) and ultrasound (US) imaging must have both high optical absorption and high echogenicity. Integrating a highly absorbing dye into the lipid shell of gas core nanobubbles (NBs) adds PA contrast to existing US contrast agents but may impact agent ultrasonic response.

Aim: We report on the development and ultrasonic characterization of lipid-shell stabilized C3F8 NBs with integrated Sudan Black (SB) B dye in the shell as dual-modal PA-US contrast agents.

Approach: Perfluoropropane NBs stabilized with a lipid shell including increasing concentrations of SB B dye were formulated by amalgamation (SBNBs). Physical properties of SBNBs were characterized using resonant mass measurement, transmission electron microscopy and pendant drop tensiometry. Concentrated bubble solutions were imaged for 8 min to assess signal decay. Diluted bubble solutions were stimulated by a focused transducer to determine the response of individual NBs to long cycle (30 cycle) US. For assessment of simultaneous multimodal contrast, bulk populations of SBNBs were imaged using a PA and US imaging platform.

Results: We produced high agent yield (∼1011) with a mean diameter of ∼200 to 300 nm depending on SB loading. A 40% decrease in bubble yield was measured for solutions with 0.3 and 0.4 mg / ml SB. The addition of SB to the shell did not substantially affect NB size despite an increase in surface tension by up to 8 mN / m. The bubble decay rate increased after prolonged exposure (8 min) by dyed bubbles in comparison to their undyed counterparts (2.5-fold). SB in bubble shells increased gas exchange across the shell for long cycle US. PA imaging of these agents showed an increase in power (up to 10 dB) with increasing dye.

Conclusions: We added PA contrast function to NBs. The addition of SB increased gas exchange across the NB shell. This has important implications in their use as multimodal agents.

Keywords: contrast agents; multimodal imaging; nanobubbles; photoacoustic imaging; rectified diffusion; ultrasound imaging.

Fig. 1

(a) Two-dimensional chemical structure of SB B and (b) optical absorption spectrum of SB B dye in different solvents (reproduced with permission).

Fig. 2

Sample size distribution of NBs and nonbuoyant particles prepared from lipid solution without SB before pore filtration as measured using the Archimedes RMM system.

Fig. 3

(a) Inverse bubble stability setup featuring a linear transducer (Toshiba, Tochigi-Ken, Japan, 6-MHz transmit, 12-MHz receive, 240 kPa peak negative pressure, M.I, 0.1), and 1% agarose mold with three narrow channels ($3\times 5\times 6\mathrm{mm}$) separated 4 mm apart; (b) US images from the inverted setup; Image of 1 in 100 diluted $0.5\mathrm{mg}/\mathrm{ml}$ SB B NBs in PBS; top of image corresponds to the top of the transducer, bottom of image corresponds to air-exposed top of channel; sample ROI indicated by yellow box; figure created with BioRender.com.

Fig. 4

Submerged multimodal US/PA experimental setup; a 10% polyacrylamide phantom with 6 hollow channels (1-mm diam; 1-mm separation) housed in a clear holder to block vessel ends; Vevo LAZR 2100 system with 256-element linear array LZ250 transducer (21 MHz central frequency; 13 to 24 MHz bandwidth) and common US/PA focus at 11-mm imaging depth. Figure created with BioRender.com.

Fig. 5

Representative transmission electron microscopy images for lipid-shelled NBs with differing SB B content (a) 0 mg; (b) 0.3; and (c) $0.5\mathrm{mg}/\mathrm{ml}$ in the lipid shell solution.

Fig. 6

(a) SBNB concentration and (b) diameter from RMM ($n=3$) differences between the average bubble diameter for varying bubble formulations were also determined to be not significant.

Fig. 7

Raw data for pendant drop tensiometry. Profiles of pendant drops of (a) PGG solution and PGG solution with (b) 0.1; (c) 0.2; (d) 0.3; (e) 0.4; (f) $0.5\mathrm{mg}/\mathrm{ml}$ added SB B, respectively, for lipid-shell stabilized NBs; ($\text{scale bar}=0.5251\mathrm{mm}$).

Fig. 8

Surface tension as measured from bulk droplets of lipid solution containing varying contributions of SB B dissolved in the lipid. ($n=20$; bars represent standard deviation).

Fig. 9

(a) US power decay normalized to maximum power of time series (Toshiba, Tochigi-Ken, Japan, 6-MHz transmit, 12 MHz receive, 240 kPa peak negative pressure, M.I, 0.1) for 1 in 100 dilution NBs in PBS with differing concentrations of SB B in the lipid shell. (b) Bubble decay rates after 8 min continuous US stimulation for lipid-stabilized with varying concentrations of SB B dye in the lipid shell.

Fig. 10

(a)–(c) Single bubble signal count as a function of acoustic pressure. ($n=3$) and SB contribution in lipid-shell. (d)–(f) Average contribution of signal types to total signal count summed over all pressures ($n=3$); average total number of signals = 511 ($0\mathrm{mg}/\mathrm{ml}$), 261 ($0.2\mathrm{mg}/\mathrm{ml}$), and 246 ($0.4\mathrm{mg}/\mathrm{ml}$).

Fig. 11

Representative PA(top)/US (bottom) images with channels containing 1:29 dilution of 0 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg Sudan Black from left to right; average PA (center; $n=9$) and US (right; $n=9$) signal from SBNBs.