Subharmonic scattering of phospholipid-shell microbubbles at low acoustic pressure amplitudes.

Subharmonic scattering of phospholipid-shell microbubbles excited at relatively low acoustic pressure amplitudes (<30 kPa) has been associated with echo responses from compression-only bubbles having initial surface tension values close to zero. In this work, the relation between sbharmonics and compression-only behavior of phospholipid-shell microbubbles was investigated, experimentally and by simulation, as a function of the initial surface tension by applying ambient overpressures of 0 and 180 mmHg. The microbubbles were excited using a 64-cycle transmit burst with a center frequency of 4 MHz and peak-negative pressure amplitudes ranging from 20 of 150 kPa. In these conditions, an increase in subharmonic response of 28.9 dB (P < 0.05) was measured at 50 kPa after applying an overpressure of 180 mmHg. Simulations using the Marmottant model, taking into account the effect of ambient overpressure on bubble size and initial surface tension, confirmed the relation between subharmonics observed in the pressure-time curves and compression-only behavior observed in the radius-time curves. The trend of an increase in subharmonic response as a function of ambient overpressure, i.e., as a function of the initial surface tension, was predicted by the model. Subharmonics present in the echo responses of phospholipid-shell microbubbles excited at low acoustic pressure amplitudes are indeed related to the echo responses from compression-only bubbles. The increase in subharmonics as a function of ambient overpressure may be exploited for improving methods for noninvasive pressure measurement in heart cavities or big vessels in the human body.

BR55: a lipopeptide-based VEGFR2-targeted ultrasound contrast agent for molecular imaging of angiogenesis.

OBJECTIVES: BR55, an ultrasound contrast agent functionalized with a heterodimer peptide targeting the vascular endothelial growth factor receptor 2 (VEGFR2), was evaluated in vitro and in vivo, demonstrating its potential for specific tumor detection. MATERIALS AND METHODS: The targeted contrast agent was prepared by incorporation of a biospecific lipopeptide into the microbubble membrane. Experiments were performed in vitro to demonstrate the binding capacities of BR55 microbubbles on immobilized receptor proteins and on various endothelial or transfected cells expressing VEGFR2. The performance of BR55 microbubbles was compared with that of streptavidin-conjugated microbubbles targeted to the same receptor by coupling them to a biotinylated antibody. The specificity of BR55 binding to human and mouse endothelial cells was determined in competition experiments with the free lipopeptide, vascular endothelial growth factor (VEGF), or a VEGFR2-specific antibody. Molecular ultrasound imaging of VEGFR2 was performed in an orthotopic breast tumor model in rats using a nondestructive, contrast-specific imaging mode. RESULTS: BR55 was shown to bind specifically to the immobilized recombinant VEGFR2 under flow (dynamic conditions). BR55 accumulation on the target over time was similar to that of microbubbles bearing a specific antibody. BR55 avidly bound to cells expressing VEGFR2, and the pattern of microbubble distribution was correlated with the pattern of receptor expression determined by immunocytochemistry. The binding of targeted microbubbles on cells was competed off by an excess of free lipopeptide, the natural ligand (VEGF) and by a VEGFR2-specific antibody (P < 0.001). Although selected for the human receptor, the VEGFR2-binding lipopeptide was also shown to recognize the rodent receptor. Tumor perfusion was assessed during the vascular phase of BR55, and then the malignant lesion was highlighted by specific accumulation of the targeted microbubbles on tumoral endothelium. The presence of VEGFR2 was confirmed by immunofluorescence staining of tumor cryosections. CONCLUSIONS: VEGFR2-targeted ultrasound contrast agents such as BR55 will likely prove useful in human for the early detection of tumors as well as for the assessment of response to specific treatments.