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.
Gas-filled nanobubbles (NBs) have been used for contrast enhanced ultrasound (US) molecular imaging similar to their microbubble (MB) counterparts. In this work we explore the use of NBs as cancer therapeutic vehicles to enhance radiation therapy and examine the role of photoacoustic (PA) imaging for quantifying the tissue response. Experiments were conducted in 58 mice bearing prostate cancer tumors. The treatment groups consisted of untreated controls (n=14), 8Gy radiation (n=14), US+MB (n=8), US+NB (n=5), US+MB+8Gy (n=12) and US+NB+8Gy (n=5). PA imaging was conducted using the VevoLAZR 21 MHz probe at pre-treatment, 2h and 24h post-treatment. To examine the changes in the tumor microenvironment post-treatment, we measured the tumor oxygen saturation (sO2), total hemoglobin (HbT) and the spectral slope (SS) as a surrogate measurement of vessel size. Histology measures of tumor vascularity/vessel size (CD31) and cellular death (TUNEL) allowed us to interpret the PA biomarker changes. The PA biomarkers revealed the following changes for the US+NB+8Gy group: (a) a 20% decrease in tumor sO2, (b) a 30% increase in tHb and (c) 20% decrease SS. These results suggest that NBs induce vessel damage from US-induced cavitation. Histology elucidates these findings: CD31 staining measuring vascularity decreases by 80% at 24h–this causes the drop in sO2 and increase in HbT; the size of vessels decreases by 10%–this is captured by the SS change. This work shows that PA imaging biomarkers can quantify these vascular changes which in turn enhance the radiation-induced cell death (up to 40%, as measured by TUNEL staining).
In this work, we examine the potential of photoacoustic imaging for understanding the biophysical mechanism of nanobubble and microbubble-based vascular disrupting therapies. We present for the first time, a direct in-vivo comparison between sub-micron nanobubbles and the commercially available microbubbles. Our results show that PA imaging of tumor oxygenation is capable of measuring the nanobubble-induced almost 40% cell death as a result of vascular disruption.
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.
In this study, we present a technique to image the enhanced particle displacement generated using an acoustic radiation force (ARF) excitation source. A swept-source OCT (SS-OCT) system with a center wavelength of 1310nm, a bandwidth of ~100nm, and an A-scan rate of 100 kHz (MEMS-VCSEL OCT Thorlabs) was used to detect gold nanoparticle (70nm in diameter) displacement .ARF was applied after the nanoparticles passed through a porous membrane and diffused into a collagen (6% collagen) matrix. B-mode, M-B mode, 3D and Speckle Variance (SV) images were acquired before and after the ARF beam was on. Differential OCT speckle variance images with and without the ARF were used to measure the particle displacement. The images were used to detect the microscopic enhancement of nanoparticle displacement generated by the ARF. Using this OCT imaging technique, the extravasation of particles though a porous membrane and characterization of the enhanced particle displacement in a collagen gel after using an ARF excitation was achieved.
In this study, multifunctional theranostic agents for photoacoustic (PA), ultrasound (US), fluorescent imaging, and for therapeutic drug delivery were developed and tested. These agents consisted of a shell made from a biodegradable Poly(lactide-co-glycolic acid) (PLGA) polymer, loaded with perfluorohexane (PFH) liquid and gold nanoparticles (GNPs) in the core, and lipophilic carbocyanines fluorescent dye DiD and therapeutic drug Paclitaxel (PAC) in the shell. Their multifunctional capacity was investigated in an in vitro study. The PLGA/PFH/DiD-GNPs particles were synthesized by a double emulsion technique. The average PLGA particle diameter was 560 nm, with 50 nm diameter silica-coated gold nano-spheres in the shell. MCF7 human breast cancer cells were incubated with PLGA/PFH/DiDGNPs for 24 hours. Fluorescent and PA images were recorded using a fluorescent/PA microscope using a 1000 MHz transducer and a 532 nm pulsed laser. For the particle vaporization and drug delivery test, MCF7 cells were incubated with the PLGA/PFH-GNPs-PAC or PLGA/PFH-GNPs particles for 6, 12 and 24 hours. The effects of particle vaporization and drug delivery inside the cells were examined by irradiating the cells with a laser fluence of 100 mJ/cm2, and cell viability quantified using the MTT assay. The PA images of MCF7 cells containing PLGA/PFH/DiD-GNPs were spatially coincident with the fluorescent images, and confirmed particle uptake. After exposure to the PLGA/PFHGNP- PAC for 6, 12 and 24 hours, the cell survival rate was 43%, 38%, and 36% respectively compared with the control group, confirming drug delivery and release inside the cells. Upon vaporization, cell viability decreased to 20%. The particles show potential as imaging agents and drug delivery vehicles.
There is currently interest in the development of nanoemulsions as imaging and therapeutic agents, particularly perfluorohexane (PFH) droplets, whose amphiphilic shell protects drugs against physico-chemical and enzymatic degradation. When delivered to their target sites, these perfluorocarbon (PFC) droplets can vaporize upon laser excitation, efficiently releasing their drug payload and/or imaging tracers. Due to the optical properties of gold, coupling PFC droplets with gold nanoparticles significantly reduces the energy required for vaporization. In this work, nanoemulsions with a PFC core and Zonyl FSP surfactant shell were produced using sonication. Droplets were characterized in terms of size and morphology using high resolution fluorescence microscopy (i.e. total internal reflection fluorescence microscopy, TIRFM), fluorescence correlation spectroscopy (FCS), transmission electron microscopy (TEM), and light scattering techniques (i.e. dynamic light scattering, DLS). The ability of PFC droplets to vaporize was demonstrated using optical light microscopy.
Phase-change contrast agents consisting of a perfluorocarbon (PFC) liquid core stabilized by a lipid, protein, or polymer shell have been proposed for a variety of clinical applications. Previous work has demonstrated that vaporization can be induced by laser irradiation through optical absorbers incorporated inside the droplet. In this study, Poly-lactide-coglycolic acid (PLGA) particles loaded with PFC liquid and silica-coated gold nanoparticles (GNPs) were developed and characterized using photoacoustic (PA) methods. Microsized PLGA particles were loaded with PFC liquid and GNPs (14, 35, 55nm each with a 20nm silica shell) using a double emulsion method. The PA signal intensity and optical vaporization threshold were investigated using a 375 MHz transducer and a focused 532-nm laser (up to 450-nJ per pulse). The laser-induced vaporization threshold energy decreased with increasing GNP size. The vaporization threshold was 850, 690 and 420 mJ/cm2 for 5μm-sized PLGA particles loaded with 14, 35 and 55 nm GNPs, respectively. The PA signal intensity increased as the laser fluence increased prior to the vaporization event. This trend was observed for all particles sizes. PLGA particles were then incubated with MDA-MB-231 breast cancer cells for 6 hours to investigate passive targeting, and the vaporization of the PLGA particles that were internalized within cells. The PLGA particles passively internalized by MDA cells were visualized via confocal fluorescence imaging. Upon PLGA particle vaporization, bubbles formed inside the cells resulting in cell destruction. This work demonstrates that GNPs-loaded PLGA/PFC particles have potential as PA theranostic agents in PA imaging and optically-triggered drug delivery systems.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.