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Biological and Environmental Phenomena at the Interface
Effect of Bubble Concentration on the In Vitro and In Vivo Performance of Highly Stable Lipid shell-stabilized Micro- and Nanoscale Ultrasound Contrast Agents Eric C. Abenojar, Pinunta Nittayacharn, Al de Leon, Reshani Perera, Yu Wang, Ilya Bederman, and Agata A. Exner Langmuir, Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019 Downloaded from http://pubs.acs.org on April 1, 2019
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Langmuir
Effect of Bubble Concentration on the In Vitro and In Vivo Performance of Highly Stable Lipid Shell-stabilized Micro- and Nanoscale Ultrasound Contrast Agents Eric C. Abenojar,1‡ Pinunta Nittayacharn,2‡ Al Christopher de Leon,1 Reshani Perera,1 Yu Wang,1 Ilya Bederman,3 Agata A. Exner1,2* 1Department
of Radiology, Case Western Reserve University, 10900 Adelbert Rd. Cleveland,
OH 44106 2Department of Biomedical Engineering, Case Western Reserve University, 10900 Adelbert Rd. Cleveland, OH 44106 3Department of Pediatrics, School of Medicine, Case Western Reserve University, 10900 Adelbert Rd. Cleveland, OH 44106
ABSTRACT Ultrasound (US) is a widely used diagnostic imaging tool because it is inexpensive, safe, portable, and broadly accessible. Ultrasound contrast agents (UCAs) are employed to enhance backscatter echo and improve imaging contrast. The most frequently utilized UCAs are echogenic bubbles made with a phospholipid or protein-stabilized hydrophobic gas core. While clinically utilized, applications of UCAs are often limited by rapid signal decay (< 5 min) in vivo under typical ultrasound imaging protocols. Here, we report on a formulation of lipid shell–stabilized perfluoropropane (C3F8) microbubbles and nanobubbles with a significantly prolonged in vivo stability. Microbubbles (875 ± 280 nm) of the target size were prepared by utilizing a multiplestep centrifugation cycle while nanobubbles (299 ± 189 nm) were isolated from the activated vial using a single centrifugation step. To provide in-depth acoustic characterization of the new construct we evaluated the effect of size and concentration on their in vitro and in vivo performance. In vitro and in vivo characterization were carried out for a range of bubble concentrations normalized by total gas volume quantified via headspace gas chromatography/mass spectrometry (GC/MS). In vitro characterization revealed that nanobubbles at different concentrations are more consistently stable over time with the highest and lowest dilutions (50fold decrease) only differing in US signal after 8 min exposure by 10.34% while for microbubbles,
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the difference was 86.46%. As expected, due to the difference in hydrodynamic diameter and scattering cross-section difference, nanobubbles showed lower overall initial signal intensity. In vivo experiments showed that both microbubbles and nanobubbles with similar initial peak signal intensity, are comparably stable over time with 66.8% and 60.6% remaining signal after 30 min, respectively. This study demonstrates that bubble concentration has significant effects on the persistence of both microbubbles and nanobubbles in vitro and in vivo, but the effects are more pronounced in larger bubbles. These effects should be taken into account when selecting the appropriate bubble parameters for future imaging applications.
Keywords: ultrasound molecular imaging, contrast agent, microbubbles, nanobubbles, lipid shellstabilized bubbles
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Langmuir
INTRODUCTION Ultrasound (US) is a widely used medical imaging modality providing real-time imaging and diagnosis. It is accessible, portable, inexpensive, safe (i.e. no ionizing radiation), and noninvasive. However, because of relatively poor soft tissue contrast compared to other imaging techniques such as magnetic resonance imaging (MRI), contrast agents have been developed to overcome this limitation and to provide a unique real-time tool to characterize tissue hemodynamic parameters.1–5 Ultrasound contrast agents (UCAs) are typically in the form of protein,3,6–8, phospholipid,9–14 or polymer8,15–20 shell-stabilized gas-filled bubbles that are highly biocompatible and echogenic.2,21–25 Among these materials, lipid shelled bubbles are desirable because they are highly biocompatible, easy to produce, and have been shown to give stronger echogenic signals.26 UCAs enhance the backscattered echo signal producing contrast arising from 1) acoustic impedance mismatch between the gas in the bubbles and the surrounding tissue or blood pool and 2) nonlinear oscillations that are highly distinct from linear tissue behavior.2 UCAs improve US image quality and offer better temporal and spatial resolution, which helps in gaining more accurate diagnosis in different applications such as cardiovascular imaging, echocardiography, quantification of blood volume, blood flow, and tissue perfusion as well as in therapeutic applications, as a drug delivery medium.3,27–29 UCAs are typically classified based on size: microbubbles (MBs, 1-8 µm) or nanobubbles (NBs,
