Radiation-force assisted targeting facilitates ultrasonic molecular imaging

Abstract

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.

Figure 1

Illustration of the effect of radiation force on targeted imaging with ultrasound. (A) Without radiation force, the majority of the contrast agents fail to contact the target site, and therefore do not bind. (B) Radiation force pushes flowing targeted contrast agents into contact with cells along a vessel wall, where they bind to target receptors.

Figure 2

Bubble resonant frequency as a function of initial radius (left axis, solid line) and size distribution of experimental bubbles (right axis, squares).

Figure 3

Instantaneous (A) radiation force, (B) velocity, and (C) displacement for a bubble of 1.0 μm radius driven by a 3-MHz, 10-cycle pulse at 14 kPa, and (D) radiation force, (E) velocity, and (F) displacement for a bubble of 1.0 μm radius driven by a 3-MHz, 10-cycle pulse at 100 kPa.

Figure 4

Time-averaged radiation force for bubbles of different sizes driven by a 3.3-μsec sinusoidal pulse at 100 kPa at their respective resonant frequencies.

Figure 5

(A) Displacement per cycle of bubbles over a range of initial radii, driven by pulses of 14 kPa at 3 and 5 MHz, and also by pulses at their respective resonant frequency. For the multimillion cycle pulses used in the experiments, the peak displacement is predicted to be on the order of mm for insonation at resonance, and thus the experimental pulse is capable of displacing the vehicle across a large vessel. (B) Displacement per cycle of bubbles over a range of initial radii, driven by pulses of 100 kPa at 3 and 5 MHz, and also by pulses at their respective resonant frequency. In this case, a pulse with a length of 100,000 cycles can produce a displacement on the order of millimeters.

Figure 6

Predicted translational velocity versus radius for microbubbles driven by 14 kPa, 1.67-sec pulses at 3 and 5 MHz.

Figure 7

Comparison between experimentally measured (black bars) and predicted (white bars) translational velocity of contrast agents during 14 kPa, 1.67-sec pulses at 3 and 5 MHz.

Figure 8

Adhesion of biotin-targeted microbubbles to avidin-coated tube with and without targeted contrast (TC) and 1.67-sec radiation force (RF) pulses at 3 MHz and 14 kPa. Two negative-targeting controls were performed: in one, the contrast microbubbles were not biotinylated; and in the other, the tube was not avidintreated. (A) Flow velocity of 6 mm/sec. (B) Flow velocity of 28 mm/sec.

Figure 9

Adherent bubbles after 1.67-sec, 14-kPa radiation force pulses in tubes with flow velocities of 6, 28, and 142 mm/sec at (A) 3 MHz and (B) 5 MHz.

Figure 10

RGD-peptide targeted bubbles adherent to α_vβ₃-expressing HUVEC cells without (left) and with (right) a 1.67-sec, 14-kPa radiation force pulse at 3 MHz. Flow velocity of 5 mm/sec.

Figure 11

Fluorescence photomicrographs showing a portion of a microvessel flow phantom. Each image is approximately 100 × 60 μm. (A) Fluorescent RGD-targeted microbubbles adherent to α_vβ₃-expressing HUVEC after 10 radiation force pulses over 100 sec. (B) Minimal adhesion of fluorescent RDG-targeted microbubbles to HUVEC is seen without application of radiation force over the same period.

Figure 12

Spectra from echoes scattered from a microvessel phantom in response to imaging pulses of (A) 440 kPa and (B) 690 kPa. Both plots show spectra from water only (dotted line), from freely flowing bubbles (thin solid line), after rinsing with water after applying radiation force pulses (thick solid line), and after rinsing with water with no radiation force application (dashed line). The narrowband spectrum of adherent bubbles after application of radiation force pulses can be observed at both pressures.