In vivo demonstration of cancer molecular imaging with ultrasound radiation force and buried-ligand microbubbles.

In designing targeted contrast agent materials for imaging, the need to present a targeting ligand for recognition and binding by the target is counterbalanced by the need to minimize interactions with plasma components and to avoid recognition by the immune system. We have previously reported on a microbubble imaging probe for ultrasound molecular imaging that uses a buried-ligand surface architecture to minimize unwanted interactions and immunogenicity. Here we examine for the first time the utility of this approach for in vivo molecular imaging. In accordance with previous results, we showed a threefold increase in circulation persistence through the tumor of a fibrosarcoma model in comparison with controls. The buried-ligand microbubbles were then activated for targeted adhesion through the application of noninvasive ultrasound radiation forces applied specifically to the tumor region. Using a clinical ultrasound scanner, microbubbles were activated, imaged, and silenced. The results showed visually conspicuous images of tumor neovasculature and a twofold increase in ultrasound radiation force enhancement of acoustic contrast intensity for buried-ligand microbubbles, whereas no such increase was found for exposed-ligand microbubbles. We therefore conclude that the use of acoustically active buried-ligand microbubbles for ultrasound molecular imaging bridges the demand for low immunogenicity with the necessity of maintaining targeting efficacy and imaging conspicuity in vivo.

Assessment of molecular imaging of angiogenesis with three-dimensional ultrasonography.

Molecular imaging (MI) with ultrasonography relies on microbubble contrast agents (MCAs) adhering to a ligand-specific target for applications such as characterizing tumor angiogenesis. It is projected that ultrasonic (US) MI can provide information about tumor therapeutic response before the detection of phenotypic changes. One of the limitations of preclinical US MI is that it lacks a comprehensive field of view. We attempted to improve targeted MCA visualization and quantification by performing three-dimensional (3D) MI of tumors expressing αvß3 integrin. Volumetric acquisitions were obtained with a Siemens Sequoia system in cadence pulse sequencing mode by mechanically stepping the transducer elevationally across the tumor in 800-micron increments. MI was performed on rat fibrosarcoma tumors (n  =  8) of similar sizes using MCAs conjugated with a cyclic RGD peptide targeted to αvß3 integrin. US MI and immunohistochemical analyses show high microbubble targeting variability, suggesting that individual two-dimensional (2D) acquisitions risk misrepresenting more complex heterogeneous tissues. In 2D serial studies, where it may be challenging to image the same plane repeatedly, misalignments as small as 800 microns can introduce substantial error. 3D MI, including volumetric analysis of inter- and intra-animal targeting, provides a thorough way of characterizing angiogenesis and will be a more robust assessment technique for the future of MI.

Improving sensitivity in ultrasound molecular imaging by tailoring contrast agent size distribution: in vivo studies.

Molecular imaging with ultrasound relies on microbubble contrast agents (MCAs) selectively adhering to a ligand-specific target. Prior studies have shown that only small quantities of microbubbles are retained at their target sites, therefore, enhancing contrast sensitivity to low concentrations of microbubbles is essential to improve molecular imaging techniques. In order to assess the effect of MCA diameter on imaging sensitivity, perfusion and molecular imaging studies were performed with microbubbles of varying size distributions. To assess signal improvement and MCA circulation time as a function of size and concentration, blood perfusion was imaged in rat kidneys using nontargeted size-sorted MCAs with a Siemens Sequoia ultrasound system (Siemans, Mountain View, CA) in cadence pulse sequencing (CPS) mode. Molecular imaging sensitivity improvements were studied with size-sorted alphavbeta3-targeted bubbles in both fibrosarcoma and R3230 rat tumor models. In perfusion imaging studies, video intensity and contrast persistence was approximately 8 times and approximately 3 times greater respectively, for "sorted 3-micron" MCAs (diameter, 3.3 +/- 1.95 microm) when compared to "unsorted" MCAs (diameter, 0.9 +/- 0.45 microm) at low concentrations. In targeted experiments, application of sorted 3-micron MCAs resulted in a approximately 20 times video intensity increase over unsorted populations. Tailoring size-distributions results in substantial imaging sensitivity improvement over unsorted populations, which is essential in maximizing sensitivity to small numbers of MCAs for molecular imaging.

Optical tracking of acoustic radiation force impulse-induced dynamics in a tissue-mimicking phantom.

Optical tracking was utilized to investigate the acoustic radiation force impulse (ARFI)-induced response, generated by a 5-MHz piston transducer, in a translucent tissue-mimicking phantom. Suspended 10-microm microspheres were tracked axially and laterally at multiple locations throughout the field of view of an optical microscope with 0.5-microm displacement resolution, in both dimensions, and at frame rates of up to 36 kHz. Induced dynamics were successfully captured before, during, and after the ARFI excitation at depths of up to 4.8 mm from the phantom's proximal boundary. Results are presented for tracked axial and lateral displacements resulting from on-axis and off-axis (i.e., shear wave) acquisitions; these results are compared to matched finite element method modeling and independent ultrasonically based empirical results and yielded reasonable agreement in most cases. A shear wave reflection, generated by the proximal boundary, consistently produced an artifact in tracked displacement data later in time (i.e., after the initial ARFI-induced displacement peak). This tracking method provides high-frame-rate, two-dimensional tracking data and thus could prove useful in the investigation of complex ARFI-induced dynamics in controlled experimental settings.

An in vivo evaluation of the effect of repeated administration and clearance of targeted contrast agents on molecular imaging signal enhancement.

Competitive inhibition diminishes ligand adhesion as receptor sites become occupied with competing ligands. It is unknown if this effect occurs in ultrasound molecular imaging studies where endothelial binding sites become occupied with adherent bubbles or bubble fragments. The goal of this pilot study was to assess the effect that repeated administration and clearance of targeted agents has on successive adhesion. Two groups of animals were imaged with 3-D ultrasonic molecular imaging. Injections and imaging were performed on Group 1 at time 0 and 60 minutes. Group 2 received injections of microbubbles at 0, 15, 30, 45 and 60 minutes with imaging at 0 and 60 minutes. At 60 minutes, Group 1 targeting relative to baseline was not significantly different from Group 2 (1.06 ± 0.27 vs. 1.08 ± 0.34, p = 0.93). Data suggest that multiple injections of targeted microbubbles do not block sufficient binding sites to bias molecular imaging data in serial studies.

Toward ultrasound molecular imaging with phase-change contrast agents: an in vitro proof of principle.

Phase-change contrast agents (PCCAs), which normally consist of nanoscale or microscale droplets of liquid perfluorocarbons in an encapsulating shell, can be triggered to undergo a phase transition to the highly echogenic gaseous state upon the input of sufficient acoustic energy. As a result of the subsequent volumetric expansion, a number of unique applications have emerged that are not possible with traditional ultrasound microbubble contrast agents. Although many studies have explored the therapeutic aspects of the PCCA platform, few have examined the potential of PCCAs for molecular imaging purposes. In this study, we demonstrate a PCCA-based platform for molecular imaging using α(v)ß(3)-targeted nanoscale PCCAs composed of low-boiling-point perfluorocarbons. In vitro, nanoscale PCCAs adhered to target cells, could be activated and imaged with a clinical ultrasound system and produced a six-fold increase in image contrast compared with non-targeted control PCCAs and a greater than fifty-fold increase over baseline. Data suggest that low-boiling-point nanoscale PCCAs could enable future ultrasound-based molecular imaging techniques in both the vascular and extravascular spaces.

Experimental validation of displacement underestimation in ARFI ultrasound.

Acoustic radiation force impulse (ARFI) imaging is an elastography technique that uses ultrasonic pulses to displace and track tissue motion. Previous modeling studies have shown that ARFI displacements are susceptible to underestimation due to lateral and elevational shearing that occurs within the tracking resolution cell. In this study, optical tracking was utilized to experimentally measure the displacement underestimation achieved by acoustic tracking using a clinical ultrasound system. Three optically translucent phantoms of varying stiffness were created, embedded with subwavelength diameter microspheres, and ARFI excitation pulses with F/1.5 or F/3 lateral focal configurations were transmitted from a standard linear array to induce phantom motion. Displacements were tracked using confocal optical and acoustic methods. As predicted by earlier finite element method studies, significant acoustic displacement underestimation was observed for both excitation focal configurations; the maximum underestimation error was 35% of the optically measured displacement for the F/1.5 excitation pulse in the softest phantom. Using higher F/#, less tightly focused beams in the lateral dimension improved accuracy of displacements by approximately 10 percentage points. This work experimentally demonstrates limitations of ARFI implemented on a clinical scanner using a standard linear array and sets up a framework for future displacement tracking validation studies.

An in vivo validation of the application of acoustic radiation force to enhance the diagnostic utility of molecular imaging using 3-d ultrasound.

For more than a decade, the application of acoustic radiation force (ARF) has been proposed as a mechanism to increase ultrasonic molecular imaging (MI) sensitivity in vivo. Presented herein is the first noninvasive in vivo validation of ARF-enhanced MI with an unmodified clinical system. First, an in vitro optical-acoustical setup was used to optimize system parameters and ensure sufficient microbubble translation when exposed to ARF. 3-D ARF-enhanced MI was then performed on 7 rat fibrosarcoma tumors using microbubbles targeted to α(v)ß3 and nontargeted microbubbles. Low-amplitude (<25 kPa) 3-D ARF pulse sequences were tested and compared with passive targeting studies in the same animal. Our results demonstrate that a 78% increase in image intensity from targeted microbubbles can be achieved when using ARF relative to the passive targeting studies. Furthermore, ARF did not significantly increase image contrast when applied to nontargeted agents, suggesting that ARF did not increase nonspecific adhesion.

Phase-change nanoparticles using highly volatile perfluorocarbons: toward a platform for extravascular ultrasound imaging.

Recent efforts using perfluorocarbon (PFC) nanoparticles in conjunction with acoustic droplet vaporization has introduced the possibility of expanding the diagnostic and therapeutic capability of ultrasound contrast agents to beyond the vascular space. Our laboratories have developed phase-change nanoparticles (PCNs) from the highly volatile PFCs decafluorobutane (DFB, bp =-2 °C) and octafluoropropane (OFP, bp =-37 °C ) for acoustic droplet vaporization. Studies with commonly used clinical ultrasound scanners have demonstrated the ability to vaporize PCN emulsions with frequencies and mechanical indices that may significantly decrease tissue bioeffects. In addition, these contrast agents can be formulated to be stable at physiological temperatures and the perfluorocarbons can be mixed to modulate the balance between sensitivity to ultrasound and general stability. We herein discuss our recent efforts to develop finely-tuned diagnostic/molecular imaging agents for tissue interrogation. We discuss studies currently under investigation as well as potential diagnostic and therapeutic paradigms that may emerge as a result of formulating PCNs with low boiling point PFCs.