We present the first study of the effects of monolayer shell physicochemical properties on the destruction of lipid-coated microbubbles during insonification with single, one-cycle pulses at 2.25 MHz and low-duty cycles. Shell cohesiveness was changed by varying phospholipid and emulsifier composition, and shell microstructure was controlled by postproduction processing. Individual microbubbles with initial resting diameters between 1 and 10 μm were isolated and recorded during pulsing with bright-field and fluorescence video microscopy. Microbubble destruction occurred through two modes: acoustic dissolution at 400 and 600 kPa and fragmentation at 800 kPa peak negative pressure. Lipid composition significantly impacted the acoustic dissolution rate, fragmentation propensity, and mechanism of excess lipid shedding. Less cohesive shells resulted in micron-scale or smaller particles of excess lipid material that shed either spontaneously or on the next pulse. Conversely, more cohesive shells resulted in the buildup of shell-associated lipid strands and globular aggregates of several microns in size; the latter showed a significant increase in total shell surface area and lability. Lipid-coated microbubbles were observed to reach a stable size over many pulses at intermediate acoustic pressures. Observations of shell microstructure between pulses allowed interpretation of the state of the shell during oscillation. We briefly discuss the implications of these results for therapeutic and diagnostic applications involving lipid-coated microbubbles as ultrasound contrast agents and drug/gene delivery vehicles.

A novel drug delivery vehicle that specifically targets using ultrasound radiation force (USRF) and biotin-avidin interactions is presented. Model vehicles consist of avidinated fluorescent nanobeads bound directly to the biotinylated lipid shells of preformed microbubbles. USRF was used to deflect the vehicle from the center of flow to a tube surface in order to facilitate receptor-ligand mediated adhesion. At wall shear stress levels commensurate with venous and arterial flow, USRF was used to direct the vehicles to a biotinylated tube surface. Subsequent high-pressure pulses fragmented the carrier, and molecular interactions induced deposition of the nanobeads on the wall. Targeting of nanobeads to the tube was molecularly specific and dependent on, in order of importance, vehicle concentration, wall shear stress, nanobead size, and insonation time. The observation that portions of the microbubble lipid monolayer shell remain attached to adherent nanobeads is important for future consideration of drug transport mechanisms. This versatile method of delivery is shown to enable targeted deposition of nanoparticles in shear flow and could be modified to carry therapeutic agents for controlled release in targeted delivery applications.

Ultrasonic molecular imaging employs contrast agents, such as microbubbles, nanoparticles, or liposomes, coated with ligands specific for receptors expressed on cells at sites of angiogenesis, inflammation, or thrombus. Concentration of these highly echogenic contrast agents at a target site enhances the ultrasound signal received from that site, promoting ultrasonic detection and analysis of disease states. In this article, we show that acoustic radiation force can be used to displace targeted contrast agents to a vessel wall, greatly increasing the number of agents binding to available surface receptors. We provide a theoretical evaluation of the magnitude of acoustic radiation force and show that it is possible to displace micron-sized agents physiologically relevant distances. Following this, we show in a series of experiments that acoustic radiation force can enhance the binding of targeted agents: The number of biotinylated microbubbles adherent to a synthetic vessel coated with avidin increases as much as 20-fold when acoustic radiation force is applied; the adhesion of contrast agents targeted to alpha(v)beta3 expressed on human umbilical vein endothelial cells increases 27-fold within a mimetic vessel when radiation force is applied; and finally, the image signal-to-noise ratio in a phantom vessel increases up to 25 dB using a combination of radiation force and a targeted contrast agent, over use of a targeted contrast agent alone.

Interest in ultrasound contrast agents (lipid-shelled microbubbles) as delivery vehicles is increasing; however, the biodistribution of these agents remains uncharacterized, both with and without ultrasound. In this study, an 18 F-labeled lipid ([ 18 F]fluorodipalmitin), incorporated in microbubble shells, was used as a dynamic microPET probe for quantitative 90-minute biodistribution measurements in male Fischer 344 rats (n = 2). The spleen retained the highest concentration of radioactive lipid at ~2.6 percent-injected dose per cubic centimeter (% ID/cc) and the liver demonstrated the largest total accumulation (~17 % ID). The microbubble pharmacokinetic profile differed from free lipid, which is rapidly cleared from blood, and liposomes, which remain in circulation. Additionally, region of interest (ROI) analysis over 60 minutes post-ultrasound treatment quantified the delivery of lipid by therapeutic ultrasound from microbubbles to kidney tissue (n = 8). The ultrasound sequence consisted of a 200 kPa, 5.3 MHz radiation force pulse followed by a 1.6 MPa, 1.4 MHz fragmentation pulse and was applied to one kidney, while the contralateral kidney served as a control. ROI-estimated activity in treated kidneys was slightly but significantly greater at 0 and 60 minutes than in untreated kidneys (p = 0.0012 and 0.0035, respectively). This effect increased with the number of microbubbles injected (p = 0.006). In summary, [ 18 F]fluorodipalmitin was used to characterize the biodistribution of contrast microbubble shells and the deposition of lipid was shown to be locally increased after insonation.

Acquisition of the epithelial-mesenchymal transition (EMT) tumor phenotype is associated with impaired chemotherapeutic delivery and a poor prognosis. In this study, we investigated the application of therapeutic ultrasound methods available in the clinic to increase nanotherapeutic particle accumulation in epithelial and EMT tumors by labeling particles with a positron emission tomography tracer. Epithelial tumors were highly vascularized with tight cell-cell junctions, compared to EMT tumors where cells displayed an irregular, elongated shape with loosened cell-cell adhesions and a reduction in E-cadherin and cytokeratins 8/18 and 19. Without ultrasound, the accumulation of liposomal nanoparticles administered to tumors in vivo was ~1.5 times greater in epithelial tumors than EMT tumors. When ultrasound was applied, both nanoaccumulation and apparent tumor permeability were increased in both settings. Notably, ultrasound effects differed with thermal and mechanical indices, such that increasing the thermal ultrasound dose increased nanoaccumulation in EMT tumors. Taken together, our results illustrate how ultrasound can be used to enhance nanoparticle accumulation in tumors by reducing their intratumoral pressure and increasing their vascular permeability.

Ultrasonic molecular imaging employs contrast agents, such as microbubbles, nanoparticles, or liposomes, coated with ligands specific for receptors expressed on cells at sites of angiogenesis, inflammation, or thrombus. Concentration of these highly echogenic contrast agents at a target site enhances the ultrasound signal received from that site, promoting ultrasonic detection and analysis of disease states. In this article, we show that acoustic radiation force can be used to displace targeted contrast agents to a vessel wall, greatly increasing the number of agents binding to available surface receptors. We provide a theoretical evaluation of the magnitude of acoustic radiation force and show that it is possible to displace micron-sized agents physiologically relevant distances. Following this, we show in a series of experiments that acoustic radiation force can enhance the binding of targeted agents: The number of biotinylated microbubbles adherent to a synthetic vessel coated with avidin increases as much as 20-fold when acoustic radiation force is applied; the adhesion of contrast agents targeted to A v B 3 expressed on human umbilical vein endothelial cells increases 27-fold within a mimetic vessel when radiation force is applied; and finally, the image signal-to-noise ratio in a phantom vessel increases up to 25 dB using a combination of radiation force and a targeted contrast agent, over use of a targeted contrast agent alone. Mol Imaging (2004) 3, 135 -148.

Objectives In ultrasound molecular imaging, a sequence of high pressure ultrasound pulses is frequently applied to destroy bound targeted microbubbles in order to quantify accumulated microbubbles or to prepare for successive microbubble injections; however, the potential for biological effects from such a strategy has not been fully investigated. Here, we investigate the effect of high pressure insonation of bound microbubbles and the potential for thrombogenic effects. Materials and methods A total of 114 mice carrying either Met-1 or NDL tumors was insonified (Siemens Sequoia system, 15L8 transducer, 5 MHz color-Doppler pulses, 4 MPa or 2 MPa peak-negative pressure, 8.1 ms pulse repetition period, 6-cycle pulse length, and 900 ms insonation). Microbubbles conjugated with cyclic RGD or LXY-3 peptides, or control (no peptide) microbubbles were injected and contrast pulse sequencing (CPS) was used to visualize the flowing and bound microbubbles. An anti-CD41 antibody was injected in a subset of animals to block potential thrombogenic effects. Results Following the accumulation of targeted microbubbles and high pressure (4 MPa) insonation, reduced blood flow, as demonstrated by a reduction in echoes from flowing microbubbles, was observed in 20 Met-1 mice (71%) and 4 NDL mice (40%). The area of low image intensity increased from 22 ± 13% to 63 ± 17% of the observed plane in the Met-1 model (p<0.01) and from 16 ± 3% to 45 ± 24% in the NDL model (p<0.05). Repeated microbubble destruction at 4 MPa increased the area of low image intensity to 76.7 ± 13.4% (p<0.05). The fragmentation of bound microbubbles with a lower peak-negative pressure (2 MPa) reduced the occurrence of the blood flow alteration to 28% (5 of 18 Met-1 tumor mice). The persistence of the observed blood flow change was approximately 30 minutes after the microbubble destruction event. Dilated vessels and enhanced extravasation of 150,000 MW FITC-dextran were observed by histology and confocal microscopy. Pre-injection of an anti-CD41 antibody blocked the reduction of tumor blood flow, where a reduction in blood flow was observed in only 1 of 26 animals. Conclusion High pressure fragmentation of microbubbles bound to tumor endothelial receptors reduced blood flow within two syngeneic mouse tumor models for ~30 minutes. Platelet activation, likely resulting from the injury of small numbers of endothelial cells, was the apparent mechanism for the flow reduction.

Gold nanoparticles (GNPs) are non-toxic, can be functionalized with ligands, and preferentially accumulate in tumors. We have developed a 13.56 MHz radiofrequency-electromagnetic field (RF-EM) delivery system capable of generating high electric field strengths required for non-invasive, non-contact heating of GNPs. The bulk heating and specific heating rates were measured as a function of NP size and concentration. It was found that heating is both size and concentration dependent, with 5 nm particles producing a 50.6±0.2°C temperature rise in 30 s for 25 μg/mL gold (125 W input). The specific heating rate was also size and concentration dependent, with 5 nm particles producing a specific heating rate of 356±78 kW/g gold at 16 μg/mL (125 W input). Furthermore, we demonstrate that cancer cells incubated with GNPs are killed when exposed to 13.56 MHz RFEM fields. Compared to cells that were not incubated with GNPs, 3 out of 4 RF-treated groups showed a significant enhancement of cell death with GNPs (p<0.05). GNP-enhanced cell killing appears to require temperatures above 50°C for the experimental parameters used in this study. Transmission electron micrographs show extensive vacuolization with the combination of GNPs and RF treatment.