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. 2017 Oct 18;7(1):13517.
doi: 10.1038/s41598-017-13741-1.

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

Christopher Hernandez  1 , Sahil Gulati  2   3 , Gabriella Fioravanti  1 , Phoebe L Stewart  4   5 , Agata A Exner  6   7
Affiliations

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

Christopher Hernandez et al. Sci Rep. .

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.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Production of crosslinked nanobubbles (CL-NBs). (A) UV irradiation is used to form a crosslinked network of the biodegradable polymer N,N-diethyl acrylamide (NNDEA) with cross-linker N,N-bis(acryoyl) cystamine (BAC). (B) Schematic diagram of a CL-NB. A P(NNDEA-co-BAC) crosslinked mesh stabilizes an outer pluronic/phospholipid layer enclosing a perfluorocarbon gas core. Illustration credit to Tiffany Yang.
Figure 2
Figure 2
Ultrasound-driven nanobubble dissipation and size characterization. (A) Schematic of the ultrasound transducer (bottom) and agarose phantom/sample location (blue). (B) Ultrasound images of crosslinked and non-crosslinked NBs before, during and after a high-power ultrasound flash cycle. (C) Dynamic light scattering intensity-weighted size distributions of crosslinked NBs before (blue) and after (red) ultrasound destruction. (D) Dynamic light scattering intensity-weighted size distributions of non-crosslinked NBs before (blue) and after (red) ultrasound destruction. Insets in panels C and D show the mean intensity-weighted hydrodynamic diameters for each group.
Figure 3
Figure 3
Cryo-EM images of CL-NBs. (A) A single CL-NB next to the carbon support layer of the EM grid (upper right-hand corner). (B) A larger nanobubble encapsulating a smaller nanobubble. (C) Two CL-NBs interacting with a larger malformed nanobubble (light center). Scale bars, 100 nm.
Figure 4
Figure 4
Cryo-EM images of sonicated CL-NBs. (A–C) Disrupted nanobubbles are shown colorized for better visualization. In panel A, multiple disrupted CL-NBs are observed along the carbon support layer of the EM grid. Occasionally, a multi-layered disrupted CL-NB is observed as in panel B. In panel C, one intact CL-NB is observed with a dense core (arrow). (D–F) Non-colorized versions of panels A-C. Scale bars, 100 nm.
Figure 5
Figure 5
Cryo-EM images of sonicated non-crosslinked nanobubbles. (A–C) Disrupted nanobubbles are shown colorized for better visualization. Typically, disrupted nanobubbles are observed along the somewhat hydrophobic carbon support layer of the EM grid (dark gray). Both deformed sheet-like structures and dot-like objects are observed suggesting the instability of nanobubbles after sonication. (D–F) Non-colorized versions of panels A-C. Scale bars, 100 nm.
Figure 6
Figure 6
Effect of extended electron beam exposure on CL-NBs. (A) Representative CL-NBs imaged with a typical electron beam exposure (60 e−/Å2) (left) and after extended electron beam exposure (900 e−/Å2) (right). Scale bars, 100 nm. (B) Density plot analyses of these CL-NBs before (blue) and after extended electron beam exposure (red). The plots reveal a decrease in the density in the center of the nanobubbles suggesting loss of perfluorocarbon gas following extended electron beam exposure. The density of the surrounding frozen ice layer before and after extended electron beam exposure has been normalized to aid comparison.
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