Acoustic Vaporization Research Articles

In Vitro and In Vivo Behavioral Evaluation of Condensed Lipid-Coated Perfluorocarbon Nanodroplets

BackgroundPhase-shift contrast agents consist of a liquid perfluorocarbon core that can be vaporized by ultrasound to generate echogenic contrast with excellent spatiotemporal control. The purpose of the present work was to evaluate the in vitro and in vivo behavior of condensed lipid-shelled nanodroplets using different analytical procedures. MethodsIn this study, perfluorobutane nanodroplets were prepared by condensation of precursor fluorescently labeled lipid shelled microbubbles and were characterized in terms of size distribution, gas core content, in vitro stability in blood as well as for their acoustic vaporization behavior using a custom-made set-up. In particular, their in vivo behavior was thoroughly investigated after intravenous bolus injection in rats. To this end, we report, for the first time, the efficient use of three complementary detection procedures to assess the NDs in vivo persistence namely 1) Ultrasound contrast imaging of vaporized NDs, 2) Gas chromatography-mass spectrometry to determine the perfluorobutane core content and 3) Fluorescence intensity measurement in the collected blood samples. ResultsThe Coulter Counter Multisizer results confirmed the size distribution shift post-condensation. Furthermore, similar PFB concentration from MBs and NDs suspensions was obtained, thus demonstrating an exceptionally low rate of microbubbles breaking and spontaneous nanodroplets vaporization. As expected, these nanoscale droplets display longer circulation time compared to clinically approved microbubbles and only slight variations of half-life were observed between the three monitoring procedures. Finally, echogenic signal observed in focal area of the liver and spleen after vaporization was confirmed by accumulation of fluorescent nanodroplets in these organs. ConclusionThese results further contribute to deepen our understanding of both in vitro and in vivo behavior of sonoresponsive nanodroplets, which is key to enable efficient clinical translation.

Droplets for acoustic vaporization: Formulations, properties and applications

The Acoustic Droplet Vaporization (ADV) (1), a phase-change of the droplets core from liquid to vapor phase upon ultrasound irradiation, burst renewed interest in droplet emulsions, a traditional topic of colloidal science, opening up innovative applications in biomedicine. Droplets undergoing ADV share similar liquid cores, typically perfluorocarbons (PFCs), however, the nature of the shells can be polymeric (2) or lipidic (3, 4). Such difference imparts to phase-change droplets diverse acoustic and mechanical behaviors (5). In this contribution we present some results concerning a general strategy for the formulation of polymer or lipid shelled submicron droplets. Other key points for the use of ADV-responsive in biomedical applications will be addressed. Recently we have extended the concepts of ADV to radiation responsive droplets for dosimetry in cancer treatment with hadronic radiation. Results on this activity will be also reported (6).

Acoustic vaporization of perfluorohexane droplets is induced by heterogeneous nucleation at 1.1 MHz

We investigated the ADV of perfluorohexane (PFH) nano and micro-droplets at a frequency of 1.1 MHz, at conditions where there is no superharmonic focusing. Our experiments were performed on suspensions of droplets in glycerol to avoid sedimentation. The ADV pressure threshold was defined as the pressure for which there is half a chance to observe a vaporization event. Our experiments showed that the threshold depends on the number of insonified droplets and the data were fitted by a statistical model, that is independant on the mechanism leading to ADV. The fit allows the derivation of the threshold in the case of a single insonified droplet. We further observed that the value of the ADV pressure threshold of a single PFH droplet decreases as its radius increases. In addition, at a constant droplet radius, the threshold did not vary when the PFH volume inside the droplet decreases, thanks to the encapsulation of an increasing number of water droplets. These results are incompatible with a model of homogeneous nucleation (where a nucleus can appear anywhere into the PFH volume). However, we developed a heterogeneous nucleation model, where the nucleus appears at the inner droplet surface, that successfully predicts our experimental ADV results.

In Vivo Ultrasound Imaging of Macrophages Using Acoustic Vaporization of Internalized Superheated Nanodroplets.

Activating patients' immune cells, either by reengineering them or treating them with bioactive molecules, has been a breakthrough in the field of immunotherapy and has revolutionized treatment, especially against cancer. As immune cells naturally home to tumors or injured tissues, labeling such cells holds promise for non-invasive tracking and biologic manipulation. Our study demonstrates that macrophages loaded with extremely low boiling point perfluorocarbon nanodroplets not only survive ultrasound-induced phase change but also maintain their phagocytic function. Unlike observations made when using higher boiling point perfluorocarbon nanodroplets, our results show that phase change occurs intracellularly at a low mechanical index using a clinical scanner operating within the energy limit set by the Food and Drug Administration (FDA). After nanodroplet-loaded macrophages were given intravenously to nude rats, they were invisible in the liver when imaged at a very low mechanical index using a clinical ultrasound scanner. They became visible when power was increased but still within the FDA limits up to 8 h after administration. The acoustic labeling and in vivo detection of macrophages using a clinical ultrasound scanner represent a paradigm shift in the field of cell tracking and pave the way for potential therapeutic strategies in the clinical setting.

Thermal and Acoustic Stabilization Of Volatile Phase-Change Contrast Agents Via Layer-By-Layer Assembly.

Phase-change contrast agents (PCCAs) are perfluorocarbon nanodroplets (NDs) that have been widely studied for ultrasound imaging in vitro, pre-clinical studies, and most recently incorporated a variant of PCCAs, namely a microbubble-conjugated microdroplet emulsion, into the first clinical studies. Their properties also make them attractive candidates for a variety of diagnostic and therapeutic applications including drug-delivery, diagnosis and treatment of cancerous and inflammatory diseases, as well as tumor-growth tracking. However, control over the thermal and acoustic stability of PCCAs both in vivo and in vitro has remained a challenge for expanding the potential utility of these agents in novel clinical applications. As such, our objective was to determine the stabilizing effects of layer-by-layer assemblies and its effect on both thermal and acoustic stability. We utilized layer-by-layer (LBL) assemblies to coat the outer PCCA membrane and characterized layering by measuring zeta potential and particle size. Stability studies were conducted by; 1) incubating the LBL-PCCAs at atmospheric pressure at 37∘C and 45∘C followed by; 2) ultrasound-mediated activation at 7.24 MHz and peak-negative pressures ranging from 0.71 - 5.48 MPa to ascertain nanodroplet activation and resultant microbubble persistence. The thermal and acoustic properties of decafluorobutane gas-condensed nanodroplets (DFB-NDs) layered with 6 and 10 layers of charge-alternating biopolymers, (LBL6NDs and LBL10NDs) respectively, were studied and compared to non-layered DFB-NDs. Half-life determinations were conducted at both 37∘C and 45∘C with acoustic droplet vaporization (ADV) measurements occurring at 23∘C. Successful application of up to 10 layers of alternating positive and negatively charged biopolymers onto the surface membrane of DFB-NDs was demonstrated. Two major claims were substantiated in this study; namely, (1) biopolymeric layering of DFB-NDs imparts a thermal stability up to an extent; and, (2) both LBL6NDs and LBL10NDs did not appear to alter particle acoustic vaporization thresholds, suggesting that the thermal stability of the particle may not necessarily be coupled with particle acoustic vaporization thresholds. Results demonstrate that the layered PCCAs had higher thermal stability, where the half-lifes of the LBLxNDs are significantly increased after incubation at 37∘C and 45∘C. Furthermore, the acoustic vaporization profiles the DFB-NDs, LBL6NDs, and LBL10NDs show that there is no statistically significant difference between the acoustic vaporization energy required to initiate acoustic droplet vaporization.

Perfluorocarbon nanodroplet size, acoustic vaporization, and inertial cavitation affected by lipid shell composition in vitro.

Perfluorocarbon nanodroplets (PFCnDs) are ultrasound contrast agents that phase-transition from liquid nanodroplets to gas microbubbles when activated by laser irradiation or insonated with an ultrasound pulse. The dynamics of PFCnDs can vary drastically depending on the nanodroplet composition, including the lipid shell properties. In this paper, we investigate the effect of varying the ratio of PEGylated to non-PEGylated phospholipids in the outer shell of PFCnDs on the acoustic nanodroplet vaporization (liquid to gas phase transition) and inertial cavitation (rapid collapse of the vaporized nanodroplets) dynamics in vitro when insonated with focused ultrasound. Nanodroplets with a high concentration of PEGylated lipids had larger diameters and exhibited greater variance in size distribution compared to nanodroplets with lower proportions of PEGylated lipids in the lipid shell. PFCnDs with a lipid shell composed of 50:50 PEGylated to non-PEGylated lipids yielded the highest B-mode image intensity and duration, as well as the greatest pressure difference between acoustic droplet vaporization onset and inertial cavitation onset. We demonstrate that slight changes in lipid shell composition of PFCnDs can significantly impact droplet phase transitioning and inertial cavitation dynamics. These findings can help guide researchers to fabricate PFCnDs with optimized compositions for their specific applications.

Acoustic droplet vaporization for nonthermal ablation of brain tumors

Acoustic vaporization of perfluorobutane-based phase-shift nanoemulsions (PSNE) can be used to nucleate inertial cavitation (IC) in vivo. The acoustic pressure amplitude must exceed a threshold for vaporization of PFB-based nanoemulsions. Two focused ultrasound transducers with a center frequency of 837 kHz were oriented such that their focal volumes overlapped and the acoustic pressure amplitude was amplified. In this study, the dual transducer system was combined with circulating PSNE to nucleate IC in established brain tumors, leading to nonthermal ablation of the tumors. For comparison, microbubble ultrasound agents (UCA) were used as IC nuclei for ultrasound-mediated tumor ablation. The ablation volume was confined to focal volume when PSNE were used to nucleate IC, whereas pre-focal damage was observed when UCA were used as IC nuclei. Additionally, PSNE-nucleated IC ablated a larger percentage of the brain tumors on average than MB-nucleated IC (89.46.8% vs 11.17.7%, respectively). These results suggest that PFB-based PSNE may be used to significantly reduce the inertial cavitation threshold in the cerebrovasculature, and when combined with transcranial focused ultrasound, enable efficient focal intracranial nonthermal ablation.

Biomimetic lung surfactant nanodrops for ultrasound imaging

Nanodrops comprising a perfluorocarbon liquid core can be acoustically vaporized into echogenic microbubbles for ultrasound imaging. Packaging the microbubble in its condensed liquid state provides some advantages, including in situ activation of the acoustic signal, longer circulation persistence, and the advent of expanded diagnostic and therapeutic applications in pathologies, which exhibit compromised vasculature. One obstacle to clinical translation is the inability of the limited surfactant present on the nanodrop to encapsulate the greatly expanded microbubble interface, resulting in ephemeral microbubbles with limited utility. In this study, we examine a biomimetic approach to stabilizing an expanding gas surface by employing the lung surfactant replacement, Beractant. Lung surfactant contains a suite of lipids and surfactant proteins that provides efficient shuttling of material from bilayer folds to the monolayer surface. We hypothesized that Beractant would improve stability of acoustically vaporized microbubbles. To test this hypothesis, we characterized Beractant surface dilation mechanics and revealed a novel biophysical phenomenon of rapid interfacial melting, spreading, and re-solidification. We then harnessed this unique spreading capability to increase the stability and echogenicity of microbubbles produced after acoustic droplet vaporization for in vivo ultrasound imaging. Such biomimetic lung surfactant-stabilized nanodrops may be useful for applications in ultrasound imaging and therapy.

An enhanced, rational model to study acoustic vaporization dynamics of a bubble encapsulated within a nonlinearly elastic shell

Acoustic droplet vaporization (ADV) is a new approach to generate vapor bubbles that have potentially broad medical applications. ADV-generated bubbles can be used as contrast agents in acoustic imaging, as drug carriers to deliver drugs to particular targets, and also in embolotherapy, thermal therapy, and histotripsy. However, despite much progress, ADV dynamics have still not been well understood and properly modeled. In this paper, we present a theoretical study of ultrasound-induced evaporation of a droplet encapsulated by a shell. The main emphasis of this theoretical study is on a proper description of the supercritical state occurring after bubble collapse. For this purpose, an isentropic equation of state for a van der Waals gas is used to describe the bubble behavior in the supercritical state. Sensitivity of the vaporization process is investigated for different acoustic and geometrical parameters and mechanical properties of the shell. Results show that the value of the minimum pressure required for direct vaporization (without any oscillatory behavior) depends on shell elasticity and initial size of the droplet, especially at high frequencies (greater than 2[MHz]). Moreover, it has been shown that applying an acoustic wave with proper phase such that thermal equilibrium of the bubble temperature with the surrounding liquid is attained, results in direct vaporization at lower acoustic pressure.

Repeated Acoustic Vaporization of Perfluorohexane Nanodroplets for Contrast-Enhanced Ultrasound Imaging.

Superheated perfluorocarbon nanodroplets are emerging ultrasound imaging contrast agents that boast biocompatible components, unique phase-change dynamics, and therapeutic loading capabilities. Upon exposure to a sufficiently high-intensity pulse of acoustic energy, the nanodroplet's perfluorocarbon core undergoes a liquid-to-gas phase change and becomes an echogenic microbubble, providing ultrasound contrast. The controllable activation leads to high-contrast images, while the small size of the nanodroplets promotes longer circulation times and better in vivo stability. One drawback, however, is that the nanodroplets can only be vaporized a single time, limiting their versatility. Recently, we and others have addressed this issue by using a perfluorohexane core, which has a boiling point above body temperature. Thus after vaporization, the microbubbles recondense back into their stable nanodroplet form. Previous work with perfluorohexane nanodroplets relied on optical activation via pulsed laser absorption of an encapsulated dye. This strategy limits the imaging depth and temporal resolution of the method. In this study, we overcome these limitations by demonstrating acoustic droplet vaporization with 1.1-MHz high-intensity focused ultrasound (HIFU). A short-duration, high-amplitude pulse of focused ultrasound provides a sufficiently strong peak negative pressure to initiate vaporization. A custom imaging sequence was developed to enable the synchronization of a HIFU transducer and a linear array imaging transducer. We show a visualization of repeated acoustic activation of perfluorohexane nanodroplets in polyacrylamide tissue-mimicking phantoms. We further demonstrate the detection of hundreds of vaporization events from individual nanodroplets with activation thresholds well below the tissue cavitation limit. Overall, this approach has the potential to result in reliable and repeatable contrast-enhanced ultrasound imaging at clinically relevant depths.

Synthesis and Characterization of Multi-Modal Phase-Change Porphyrin Droplets

Phase-change droplets are a class of ultrasound contrast agents that can convert into echogenic microbubbles in situ with the application of sufficient acoustic energy. Droplets are smaller and more stable than their microbubble counterparts. However, traditional ultrasound contrast agents are not trackable beyond acoustic feedback measurements, which makes quantifying contrast agent bio-distribution or accumulation ex vivo difficult. Researchers may have to rely on fluorescent or optically absorbent companion diagnostic particles to infer bio-distribution. The purpose of this protocol is to detail steps for creating multi-modal phase-change porphyrin droplets using a condensation method. Porphyrins are fluorescent molecules with distinct absorbance bands that can be conjugated onto lipids and incorporated into droplets to extend droplet versatility, enabling more robust bio-distribution while retaining acoustic properties. Seven formulations with varying porphyrin-lipid and base lipid contents were made to investigate microbubble and droplet size distributions. Characterizations suited to porphyrin-containing structures are also described in the protocol to demonstrate their analytic versatility in-solution. Sizing showed that the post-condensed mean diameters were 1.72 to 2.38 times smaller than precursor populations. Absorbance characterization showed intact assemblies had a Q-band peak of 700 nm while disrupted samples had an absorbance peak at 671 nm. Fluorescence characterization showed intact 30% porphyrin-lipid assemblies were fluorescently quenched (>97%), with fluorescence recovery achieved upon disruption. Acoustic vaporization showed that porphyrin droplets were non-echogenic at lower pressures and could be converted into echogenic microbubbles with sufficient pressures. These characterizations show the potential for porphyrin droplets to eliminate the need for absorbance or fluorescence-based companion diagnostic strategies to quantify ultrasound contrast agent bio-distribution for delivery or therapeutic applications in vivo or ex vivo.

Synthesis and Characterization of Multi-Modal Phase-Change Porphyrin Droplets.

Phase-change droplets are a class of ultrasound contrast agents that can convert into echogenic microbubbles in situ with the application of sufficient acoustic energy. Droplets are smaller and more stable than their microbubble counterparts. However, traditional ultrasound contrast agents are not trackable beyond acoustic feedback measurements, which makes quantifying contrast agent bio-distribution or accumulation ex vivo difficult. Researchers may have to rely on fluorescent or optically absorbent companion diagnostic particles to infer bio-distribution. The purpose of this protocol is to detail steps for creating multi-modal phase-change porphyrin droplets using a condensation method. Porphyrins are fluorescent molecules with distinct absorbance bands that can be conjugated onto lipids and incorporated into droplets to extend droplet versatility, enabling more robust bio-distribution while retaining acoustic properties. Seven formulations with varying porphyrin-lipid and base lipid contents were made to investigate microbubble and droplet size distributions. Characterizations suited to porphyrin-containing structures are also described in the protocol to demonstrate their analytic versatility in-solution. Sizing showed that the post-condensed mean diameters were 1.72 to 2.38 times smaller than precursor populations. Absorbance characterization showed intact assemblies had a Q-band peak of 700 nm while disrupted samples had an absorbance peak at 671 nm. Fluorescence characterization showed intact 30% porphyrin-lipid assemblies were fluorescently quenched (>97%), with fluorescence recovery achieved upon disruption. Acoustic vaporization showed that porphyrin droplets were non-echogenic at lower pressures and could be converted into echogenic microbubbles with sufficient pressures. These characterizations show the potential for porphyrin droplets to eliminate the need for absorbance or fluorescence-based companion diagnostic strategies to quantify ultrasound contrast agent bio-distribution for delivery or therapeutic applications in vivo or ex vivo.

Ultrafast dynamics of acoustic vaporization and payload release of phase-shift emulsions

Acoustic droplet vaporization (ADV) is the phase-transitioning of perfluorocarbon emulsions via focused ultrasound. ADV has been utilized in many biomedical applications. For drug delivery and tissue regeneration, we developed composite hydrogel scaffolds, termed acoustically responsive scaffolds (ARSs), which here contained fluorescently labeled monodispersed emulsions (Ø: 12.3 ± 0.8 μm). Using ultra-high speed microscopy, we investigated the dynamics of ADV (2.5 MHz, P-: 6 MPa, pulse length: 6μs) in ARSs containing emulsions with perfluoropentane, perfluorohexane, and perfluorooctane. Vaporization and growth dynamics were studied in micro- to millisecond time scales at frame rates up to 10 Mfps. During ADV, the generated bubbles expanded 4.8-, 2.8-, and 2-fold greater than the initial diameter of the perfluoropentane, perfluorohexane, and perfluorooctane emulsions, respectively. Stable bubbles formed from ADV in perfluoropentane and perfluorohexane, though growth rates post-ADV were significantly different. Bubbles recondensed within perfluorooctane emulsions, which exhibited a 20% decrease in diameter following five repeated ADV events. Stable or transient bubble formation was further compared with classical nucleation theory predictions. Additionally, we compared the release dynamics of fluorescently labeled payloads from the emulsions. The results provide physical insight enabling the modulation of bubble dynamics with ADV and hence release kinetics, which can be used for therapeutic applications.

Acoustic vaporization of internalized superheated perfluorocarbon nanodroplets for imaging and macrophage function modulation

Ultrasound (US) can detect and interact with its contrast agents, allowing perfluorocarbon-based nanodroplets (NDs) or microbubbles (MBs) to detect and treat tumors in deep tissues. Since NDs given IV passively accumulate in tumor macrophages, we aimed to determine whether phagocytosed perfluorobutane (PFB) NDs that boil at -2 °C, can be vaporized in vivo using a clinical scanner. Should that be possible, PFB NDs can then be used to deliver drugs or genes to modulate macrophage function. We formulated stable PFB NDs and validated their internalization into THP-1 macrophages using confocal microscopy. PFB ND loaded macrophages were then infused into nude rats and US imaging (low MI US imaging, 18H6 transducer) of the liver and spleen performed in vivo using a Siemens Sequoia clinical scanner before and after applying MB destruction pulses (at 1.4 MI, 10L4 transducer). PFB ND-loaded macrophages were not visible using low MI imaging, before applying MB destruction pulses. They immediately vaporized into US visible microbubbles when destruction pulses were applied over the liver and remained visible for many minutes after vaporization. Our current focus is to assess ND-loaded macrophage function before and after ND activation to determine the feasibility of modulating immune function. [Study supported by CPRIT RR150010. RFM is a CPRIT Established Investigator. Siemens ACUSON Sequoia Ultrasound System provided as a loan by Siemens Medical Solutions, USA.]

Investigation of the Acoustic Vaporization Threshold of Lipid-Coated Perfluorobutane Nanodroplets Using Both High-Speed Optical Imaging and Acoustic Methods

A combination of ultrahigh-speed optical imaging (5 × 106 frames/s), B-mode ultrasound and passive cavitation detection was used to study the vaporization process and determine both the acoustic droplet vaporization (ADV) and inertial cavitation (IC) thresholds of phospholipid-coated perfluorobutane nanodroplets (PFB NDs, diameter = 237 ± 16 nm). PFB NDs have not previously been studied with ultrahigh-speed imaging and were observed to form individual microbubbles (1–10 μm) within two to three cycles and subsequently larger bubble clusters (10–50 μm). The ADV and IC thresholds did not statistically significantly differ and decreased with increasing pulse length (20–20,000 cycles), pulse repetition frequency (1–100 Hz), concentration (108–1010 NDs/mL), temperature (20°C–45°C) and decreasing frequency (1.5–0.5 MHz). Overall, the results indicate that at frequencies of 0.5, 1.0 and 1.5 MHz, PFB NDs can be vaporized at moderate peak negative pressures (<2.0 MPa), pulse lengths and pulse repetition frequencies. This finding is encouraging for the use of PFB NDs as cavitation agents, as these conditions are comparable to those required to achieve therapeutic effects with microbubbles, unlike those reported for higher-boiling-point NDs. The differences between the optically and acoustically determined ADV thresholds, however, suggest that application-specific thresholds should be defined according to the biological/therapeutic effect of interest.

Contrast-Enhanced Sonography with Biomimetic Lung Surfactant Nanodrops.

Nanodrops comprising a perfluorocarbon liquid core can be acoustically vaporized into echogenic microbubbles for ultrasound imaging. Packaging the microbubble in its condensed liquid state provides some advantages, including in situ activation of the acoustic signal, longer circulation persistence, and the advent of expanded diagnostic and therapeutic applications in pathologies which exhibit compromised vasculature. One obstacle to clinical translation is the inability of the limited surfactant present on the nanodrop to encapsulate the greatly expanded microbubble interface, resulting in ephemeral microbubbles with limited utility. In this study, we examine a biomimetic approach to stabilize an expanding gas surface by employing the lung surfactant replacement, beractant. Lung surfactant contains a suite of lipids and proteins that provide efficient shuttling of material from bilayer folds to the monolayer surface. We hypothesized that beractant would improve stability of acoustically vaporized microbubbles. To test this hypothesis, we characterized beractant surface dilation mechanics and revealed a novel biophysical phenomenon of rapid interfacial melting, spreading, and resolidification. We then harnessed this unique functionality to increase the stability and echogenicity of microbubbles produced after acoustic droplet vaporization for in vivo ultrasound imaging. Such biomimetic lung surfactant-stabilized nanodrops may be useful for applications in ultrasound imaging and therapy.

Numerical investigation of acoustic vaporization threshold of microdroplets

A numerical model is presented for the acoustic vaporization threshold of a dodecafluoropentane (or perfluoropentane) microdroplet. The model is based on the Rayleigh-Plesset equation and is improved by properly treating the supercritical state that occurs when a bubble collapses rapidly and by employing the van der Waals equation of state to consider the supercritical state. The present computations demonstrate that the microdroplet vaporization behavior depends intricately on bubble compressibility, liquid inertia and phase-change heat transfer under acoustic excitation conditions. We present acoustic pressure-frequency diagrams for bubble growth regimes and the ADV threshold conditions. The effects of acoustic parameters, fluid properties and the droplet radius on the ADV threshold are investigated.

Lipid Shell Retention and Selective Binding Capability Following Repeated Transient Acoustic Microdroplet Vaporization.

Targeted therapy and molecular imaging using ultrasound have been widely explored using microbubble contrast agents, and more recently, activatable droplet contrast agents that vaporize when exposed to focused ultrasound have been explored. These droplets are coated with a stabilizing, functionalizable shell, typically comprised of fully saturated phospholipids. While the shedding of the lipid shell under ultrasound exposure is a well-studied phenomenon in microbubbles, it has not been fully explored in droplet-based contrast agents, particularly in those that undergo a reversible phase change and recondense following vaporization. Here, we investigate the retention of the lipid shell following repeated transient vaporization events. Two separate fluorescent markers were used to track individual lipid subpopulations: PEGylated lipids, to which targeting ligands are typically bound, and non-PEGylated lipids, which primarily contribute to droplet stability. Following confirmation of the homogeneous surface distribution of each subpopulation of shell lipids using confocal microscopy, high-speed optical imaging provided visual evidence of the ability to repeatedly induce vaporization and recondensation in micron-scale droplets using 5.208 MHz, 3.17 MPa focused ultrasound pulses transmitted from an imaging transducer. Flow cytometry analysis indicated that while PEGylated lipids were fully retained following repeated transient phase change events, 20% of the bulk lipids were shed. While this likely contributed to an observed significant reduction in the average droplet diameter, the selective binding capabilities of droplets functionalized with an RGD peptide, targeted to the integrin αvβ3, were not affected. These results indicate that repeated droplet activation may promote shifts in the droplet size distribution but will not influence the accuracy of targeting for therapy or molecular imaging.

Numerical investigation of acoustically-triggered droplet vaporization in a tube

Acoustically-triggered droplet vaporization (ADV) in a tube is numerically studied by extending a level-set method to determine the bubble-droplet interface as well as the droplet-ambient liquid interface and to include droplet evaporation and bubble compressibility effects. The Rayleigh-Plesset equation for ADV in a tube is improved by correctly evaluating the transition from spherical to tube flows. The computations of ADV show that the liquid pressure variation along the tube caused by droplet vaporization and bubble condensation is an important factor determining bubble survival and growth. The influence of tube radius on bubble growth in ADV is quantified in various acoustic amplitude and frequency conditions. Computations are also performed to investigate bubble-bubble interaction in acoustic vaporization of two droplets.

Fluorous-phase iron oxide nanoparticles as enhancers of acoustic droplet vaporization of perfluorocarbons with supra-physiologic boiling point

Perfluorocarbon emulsion nanodroplets containing iron oxide nanoparticles (IONPs) within their inner perfluorohexane (PFH) core were prepared to investigate potential use as an acoustically activatable ultrasound contrast agent, with the hypothesis that incorporation of IONPs into the fluorous phase of a liquid perfluorocarbon emulsion would potentiate acoustic vaporization. IONPs with an oleic acid (OA) hydrophobic coating were synthesized through chemical co-precipitation. To suspend IONP in PFH, OA was exchanged with perfluorononanoic acid (PFNA) via ligand exchange to yield fluorophilic PFNA-coated IONPs (PFNA-IONPs). Suspensions with various amounts of PFNA-IONPs (0–15% w/v) in PFH were emulsified in saline by sonication, using 5% (w/v) egg yolk phospholipid as an emulsifier. PFNA-IONPs were characterized with transmission electron microscopy (TEM), transmission electron cryomicroscopy (cryoTEM), and thermogravimetric analysis (TGA) with Fourier transform infrared spectroscopy (FTIR). IONP were between 5 and 10 nm in diameter as measured by electron microscopy, and hydrodynamic size of the PFH nanodroplets were 150 to 230 nm as measured by dynamic light scattering (DLS). Acoustic droplet vaporization of PFH nanodroplets (PFH-NDs) was induced using conversion pulses (100 cycle at 1.1 MHz and 50% duty cycle) provided by a focused ultrasound transducer, and formed microbubbles were imaged using a clinical ultrasound scanner. The acoustic pressure threshold needed for PFH-NDs vaporization decreased with increasing temperature and IONP content. PFH-NDs containing 5% w/v IONP converted to microbubbles at 42 °C at 2.18 MI, which is just above the exposure limits of 1.9 MI allowed by the FDA for clinical ultrasound scanners, whereas 10 and 15% emulsion vaporized at 1.87 and 1.24 MI, respectively. Furthermore, 5% IONP-loaded PFH-NDs injected intravenously into melanoma-bearing mice at a dose of 120 mg PFH/kg, converted into detectable microbubbles in vivo 5 h, but not shortly after injection, indicating that this technique detects NDs accumulated in tumors.