In this study, we evaluated if successive injections of targeted microbubbles would re- duce the ability of future injections of targeted bubbles to adhere to endothelial receptor ligands. While the scope of this study was limited to a short time period as well as a spe- cific tumor model and a specific microbubble dose, the results are encouraging. Given the dose of cRGD-targeted microbubbles injected (which was appropriate for achieving sufficient molecular imaging signal) and the inherent variability of the molecular imag- ing procedure, there was no discernible statistical difference between injecting twice and injecting five times. Ultimately, this suggests that the quantity of over-expressed αvβ3 biomarkers present must be sufficiently greater than the available ligands so as
not to present competitive inhibition. Thus, preliminary data suggests that serial mul- tiple injections in a single USMI study do not bias or compromise endothelial retention of targeted microbubbles due to competitive inhibition from prior bound bubbles or bubble fragments. What our study does not address is whether or not the adhesion of targeted bubble ligands affects the tumor biology as might be anticipated with large doses. Furthermore, we observed a slight increase in molecular targeting in both imag- ing models, the mechanism for which is still unknown. Future studies beyond this pilot should include larger animal numbers, different doses of contrast, different ligands, and longer time points.
CHAPTER 8
Improving Sensitivity in Ultrasonic Molecular Imaging
8.1 Introduction
For traditional USMI applications, backscatter intensity is relatively weak owing to the small populations retained during targeting [56; 106; 107; 108; 109; 45]. This poor binding efficiency necessitates signal amplification in USMI applications, thus providing the motivation to maximize the sensitivity to bound MCAs [56; 76; 51].
Owing to the small percentage of bound MCAs at their target sites, research in USMI has focused mainly on improvement of the contrast sensitivity through improved ligands and adhesion schemes, detection methods, and contrast delivery mechanisms [99; 110; 56; 51; 106; 48; 111; 112; 113; 114]. However, over the last several years, more attempts have been made to improve contrast sensitivity by optimizing the echogenicity of the contrast agents themselves.
In order to produce the most effective acoustic backscatter, the scattering cross- section of the microbubble must be as large as possible. In accordance with the Rayleigh scattering model, theory predicts an increase in US backscatter intensity as a function of the microbubble scattering cross-section and therefore size:
c
2010 BC Decker Inc. (mi.deckerpublishing.com) Portions reprinted, from JE Streeter, RC Gess- ner, I Miles, and PA Dayton. “Improving Sensitivity in Ultrasound Molecular Imaging by Tailoring Contrast Agent Size Distribution: In VivoStudies” Mol Imaging, 2010 Mar/Apr; 9(2): 87-95.
I = Ioσ
4πz2 (8.1)
where Io is incident intensity, σ is the microbubble scattering cross-sectional area
with a 6th order dependence on radius, and z is the distance between the transducer
and the microbubble [36].
Like a mechanical system, the microbubble’s competing balance of forces can result in a resonant frequency as it oscillates in an sinusoidal acoustic field. Thus, at the resonant frequency, the microbubble has its largest cross-sectional area and produces the most effective backscatter intensity. Based on the knowledge that microbubble size and resonant frequency are intricately coupled, Talu and colleagues proposed that in- creasing the monodispersity of a microbubble population to match the fixed frequency output of an imaging system may improve contrast imaging sensitivity [106]. Moreover, in anin vitro study of the acoustic response of monodisperse contrast agents, Kaya and colleagues determined that signal amplitude could be increased both by matching the imaging frequency to the bubbles’ resonant frequency and by increasing the diameter of the microbubbles [105]. Finally, Sirsi and colleagues recently demonstrated improve- ments in contrast to tissue ratio in the mouse kidney using high-frequency contrast imaging by increasing the mean diameter of the microbubble population [42]. Because of the significance of microbubble size in the acoustic response, recent interest has involved new production and sorting methods for MCAs, including centrifugation tech- niques, microfluidics, and electrohydrodynamic atomization [39; 41; 40; 115; 116; 117]. In addition to echogenicity, the bloodstream persistence of a microbubble is directly correlated to the initial radius of a MCA by the dissolution behavior governing lipid- shelled microbubbles. Also, accumulation of targeted MCAs in USMI experiments is generally affected by circulation persistence [118; 119]. It should be noted, however, that bloodstream persistence is a complex physiologic phenomenon that depends on
other environmental factors that are not discussed in this article.
The contrast agent currently approved by the Food and Drug Administration (FDA), DefinityR, has a polydisperse distribution with a mean diameter of around 1µm (diam- eter 1.0± 0.83 µm, as tested in this study) and a concentration of ∼1 x 1010 #
mL [120].
This distribution is characteristic of many types of lipid-shelled microbubbles, which are formed by sonic or mechanical agitation, two common formulation techniques for tar- geted and non-targeted MCAs. For standard perfusion imaging, MCA size distribution has not been a limitation, because billions of microbubbles are typically administered intravascularly, thus providing plenty of image contrast. However, for USMI applica- tions, relatively small populations of adherent MCAs remain, and thus, provide weak backscatter intensity, which ultimately limits imaging sensitivity [56; 106; 108]. In this chapter, we demonstrate in vivo that contrast sensitivity improvement in USMI and perfusion imaging applications can be achieved by increasing the mean diameter in microbubble populations.