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.

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.

Phase-change contrast agents (PCCAs), which normally consist of nano/microscale droplets of liquid perfluorocarbons (PFCs) 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 αvβ3-targeted nanoscale PCCAs composed of low-boiling-point PFCs. 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 to 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 space.

Molecular imaging (MI) with ultrasound 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 attempt to improve targeted MCA visualization and quantification by performing 3-D MI of tumors expressing αvβ3. Volumetric acquisitions were obtained with a Siemens Sequoia system in CPS 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 αvβ3. US MI and immunohistochemical analyses show high microbubble targeting variability, suggesting that individual 2-D acquisitions risk misrepresenting more complex heterogeneous tissues. In 2-D serial studies, where it may be challenging to image the same plane repeatedly, misalignments as small as 800 microns can introduce substantial error. 3-D 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.