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.
Nanobubbles are a new class of ultrasound contrast agents. Unlike conventional microbubbles, their sub-micron (~200nm) diameter allows them to extravasate outside the vasculature and accumulate in the tumor interstitium. In this study, nanobubbles with shells loaded with Sudan Black (BNB) and DiD fluorescent dye were synthesized. These nanobubbles can be used to simultaneously enhance ultrasound and photoacoustic signals for in vivo breast tumor imaging.
The nanobubbles consisted of lipid shells with a C3F8 gas core and were formed via self-assembly driven by mechanical agitation and size isolation via centrifugation. Herceptin antibody was conjugated to the BNB for targeting HER2-positive cells via standard EDC/NHS coupling chemistry. Human breast cancer cell lines (BT474 as HER2-positive and MDA-MB-231 as HER2-negative) were inoculated in the flanks of BALB/c-B17-Scid mice. Ultrasound and photoacoustic imaging (VevoLAZR, 21MHz, 720nm) were performed pre-injection and post-injection of the Herceptin conjugated BNB. The impact of Herceptin targeting was assessed by computing the PA frequency spectra and the non-linear contrast US images of the tumor regions.
Photoacoustic images of the HER2-positive tumor showed an average of 6 dB increase in contrast signal 2 mins post-injection, while the HER2-negative MDA tumors showed a negligible change in image contrast, suggesting increased uptake of Herceptin labelled BNBs. The enhanced contrast is also confirmed by the non-linear contrast signals between positive and negative tumors. The photoacoustic technique can potentially be used to examine the kinetics of BNB extravasation. This work shows the potential of BNBs as multi-modal contrast agents capable of specialized tumor imaging in vivo.
We use a novel acoustic-based flow cytometer to detect individual nanobubbles flowing in a microfluidic channel using high-frequency ultrasound and photoacoustic waves. Each individual nanobubble (or cluster of nanobubbles) flowing through the foci of high-frequency ultrasound (center frequency 375 MHz) and nanosecond laser (532 nm) pulses interacts with both pulses to generate ultrasound backscatter and photoacoustic waves. We use in-house generated nanobubbles, made of lipid shells and octafluoropropane gas core, to detect ultrasound backscatter signals using an acoustic flow cytometer. Nanobubble solutions sorted in size through differential centrifugation are diluted to 1:10,000 v/v in phosphate buffered saline solution to maximize the probability that the detected signals are from individual nanobubbles. Nanobubble populations were sized using resonant mass measurement. Results show that the amplitude of the detected ultrasound backscatter signal is dependent on the nanobubble size. The average amplitude of the ultrasound backscatter signals from at least 950 nanobubbles with an average diameter of 150 nm, 225 nm, and 350 nm was 5.1±2.5 mV, 5.3±2.3 mV, and 6.4±1.8 mV, respectively. Similarly, we detected interleaved ultrasound backscatter and photoacoustic signals from nanobubbles tagged with Sudan Black B dye. The average amplitude of the ultrasound backscatter and photoacoustic signals from these black nanobubbles with an average diameter of 238 nm is 10±11 mV and 54±75 mV, respectively. The presence of the dye on the shell suppressed unique features seen in the ultrasound backscatter from the nanobubbles without dye. At present, there is no robust commercial technique able to analyze the ultrasonic response of individual nanobubbles. The acoustic flow cytometer can potentially be used to analyze physical parameters, such as size and ultrasonic response, of individual nanobubbles.
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