Cryo-EM Visualization of Lipid and Polymer-Stabilized Perfluorocarbon Gas Nanobubbles - A Step Towards Nanobubble Mediated Drug Delivery

Abstract

Gas microbubbles stabilized with lipids, surfactants, proteins and/or polymers are widely used clinically as ultrasound contrast agents. Because of their large 1–10 µm size, applications of microbubbles are confined to the blood vessels. Accordingly, there is much interest in generating nanoscale echogenic bubbles (nanobubbles), which can enable new uses of ultrasound contrast agents in molecular imaging and drug delivery, particularly for cancer applications. While the interactions of microbubbles with ultrasound have been widely investigated, little is known about the activity of nanobubbles under ultrasound exposure. In this work, we demonstrate that cryo-electron microscopy (cryo-EM) can be used to image nanoscale lipid and polymer-stabilized perfluorocarbon gas bubbles before and after their destruction with high intensity ultrasound. In addition, cryo-EM can be used to observe electron-beam induced dissipation of nanobubble encapsulated perfluorocarbon gas.

Initial growth dynamics of 10 nm nanobubbles in the graphene liquid cell

“…The gaseous bubbles in a liquid have been used in broad such as the controlling of protein conformations (Liu et al 2005;Zhou et al 2004;Zhang et al 2013;Okumura and Itoh 2014), the drug delivery studies (Lukianova-Hleb et al 2012;Li et al 2015;Hernandez et al 2017), the direct transfer of large-size graphene films with decreased defects (Gao et al 2014;Guo et al 2018), and the performance of chemical mechanical polishing (Tsai et al 2007;Zhang et al 2016aZhang et al , b, 2018. For example, a sudden formation of nitrogen nanobubbles might induce the unfolding of protein in our body and has been associated with the origin of 'Diver's disease' .…”

Sink or float? Characterization of shell-stabilized bulk nanobubbles using a resonant mass measurement technique

“…Traditional characterization techniques such as dynamic light scattering (DLS), electro-impedance volumetric zone sensing (Coulter counter), and nanoparticle tracking analysis are unable to differentiate between gas-filled nanobubbles and similarly sized solid contaminates, leading to inaccurate/misleading bubble concentrations and size measurements 13–15. Imaging techniques such as atomic force microscopy (AFM) or electron microscopy (EM) are expensive, sample dependent, and involve processing steps that inhibit them from directly visualizing fragile nanobubbles 16–18…”

“…Nanobubble exposure to high power ultrasound disrupts the lipid shell membrane, leading to a loss of gas and consequently a decrease in the ultrasound signal. We have demonstrated that the remnant bubble shells reassemble into lipid sheets, liposomes, and micelles 18. To investigate these effects, we used RMM to characterize the structure of the nanobubbles before and after their exposure to high-intensity US.…”

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

“…The in vitro echogenicity of NBs and MBs was determined for six concentrations of bubbles with an equivalent gas volume concentration. The bubble solutions were placed in a custom-made 1.5% (w/v) agarose mold with a triple channel (L × W × H per channel = 5 × 3 × 6 mm) [44]. The agarose phantom was fixed over a 12 MHz linear array transducer and imaged using a clinical ultrasound scanner (AplioXG SSA-790A, Toshiba Medical Imaging Systems, Otawara-Shi, Japan).…”