Tailoring the size distribution of ultrasound contrast agents: possible method for improving sensitivity in molecular imaging

Abstract

Encapsulated microbubble contrast agents incorporating an adhesion ligand in the microbubble shell are used for molecular imaging with ultrasound. Currently available microbubble agents are produced with techniques that result in a large size variance. Detection of these contrast agents depends on properties related to the microbubble diameter such as resonant frequency, and current ultrasound imaging systems have bandwidth limits that reduce their sensitivity to a polydisperse contrast agent population. For ultrasonic molecular imaging, in which only a limited number of targeted contrast agents may be retained at the site of pathology, it is important to optimize the sensitivity of the imaging system to the entire population of contrast agent. This article presents contrast agents with a narrow size distribution that are targeted for molecular imaging applications. The production of a functionalized, lipid-encapsulated, microbubble contrast agent with a monodisperse population is demonstrated, and we evaluate parameters that influence the size distribution and demonstrate initial acoustic testing.

Figure 1

Overlap between resonant frequencies in a microbubble population and the −6 dB bandwidth of a 3.8 MHz 80% bandwidth transducer. A, Microbubble population similar to commercially available lipid-encapsulated contrast agents (2.1 ± 1.1 μm). B, “Nearly monodisperse” microbubble population in which the mean diameter has a resonant frequency that matches the transducer center frequency, with a standard deviation of 5% of the mean diameter.

Figure 2

Size distribution of contrast agents manufactured in our laboratory (3.7 μm with a standard deviation of 0.2 μm in this case) in contrast to the commercially available contrast agent Definity (2.7 μm with a standard deviation of 2.0 μm). The scale bar represents 10 μm.

Figure 3

Size dependence of microbubbles on gas pressure and the bulk liquid viscosity (increased with glycerine and propylene glycol [GP]) by keeping the liquid flow rate constant (30 μL/min).

Figure 4

Microscope images of microbubbles recorded at different times illustrating stability: A, t = 0; B, t = 2 hours; C, t = 4 hours; D, t = 8 hours. The microbubbles have a mean diameter of 3.7 μm with a standard deviation of 0.2 μm. The scale bar represents 10 μm.

Figure 5

A and B, Images of a population of lipid-coated monodisperse microbubbles incorporating DiI, illustrating the lipid and emulsifier phase coexistence. The scale bar represents 10 μm. C, Biotin-functionalized monodisperse microbubbles made without DiI, incorporating fluorescent avidin. Scale bar represents 5 μm.

Figure 6

Monodisperse RGD-targeted microbubbles adherent to an α_vβ₃-expressing A375m cell monolayer. Scale bar represents 5 μm.

Figure 7

A, The amplitude of scattered echoes from individual mono-disperse microbubbles had less standard deviation than polydisperse microbubble echoes. B, Echoes of individual microbubbles were more correlated for monodisperse micro-bubbles compared with polydisperse microbubbles. RMS = root mean square.