Effect of Bubble Concentration on the in Vitro and in Vivo Performance of Highly Stable Lipid Shell-Stabilized Micro- and Nanoscale Ultrasound Contrast Agents

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 multiple-step 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 (50-fold decrease) only differing in US signal after 8 min exposure by 10.34%, while for microbubbles 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.

Nanobubble Mediated Gene Delivery in Conjunction With a Hand-Held Ultrasound Scanner

“…Previous studies have suggested that altering the MB shell compositions is of great importance for the purpose of increasing the efficacy of drug delivery (Dicker et al, 2013;Abenojar et al, 2019). MB shells that have targeting ligands are especially attractive from the viewpoint of concentrating functional genes to a specific site for gene therapy.…”

Theoretical and Experimental Gas Volume Quantification of Micro- and Nanobubble Ultrasound Contrast Agents

“…Nanobubbles with similar gas volumes to commercially available micron-sized UCAs, Definity and Optison, were shown to be more reliable for BBB opening, which could be a result of its smaller size and larger number of bubbles present [28]. A recent report by our group showed that at similar gas volumes, nanobubbles are less affected by changes in gas volume concentration compared to microbubbles [25].…”

“…Several reports have used total bubble gas volume as the main parameter in evaluating bubbles with the same shell components for ultrasound activity rather than the bubble count [23][24][25]. Microbubbles of different sizes evaluated with a similar gas volume were reported to show a similar circulation half-life, which suggests that gas volume may be a better predictor of UCA stability than bubble size [26].…”

“…It is also important to note that none of these techniques are capable of distinguishing between buoyant (bubble) and non-buoyant populations of a sample, limiting their accuracy in providing accurate bubble concentrations [33]. To overcome these drawbacks, a resonant mass measurement (RMM) technique has been used to quantify the size and concentration of the bubbles separately from non-buoyant particles [25,[33][34][35][36][37] and to determine the theoretical gas volume in a nanobubble population. However, these theoretical calculations are rarely verified experimentally, despite their importance in evaluating and comparing bubbles.…”

“…However, these theoretical calculations are rarely verified experimentally, despite their importance in evaluating and comparing bubbles. Actual gas volume measurements using headspace gas chromatography/mass spectrometry (GC/MS) have rarely been performed [38] or correlated with theoretical measurements [25].…”