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

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.

[1]  Pan Li,et al.  Nanobubble-Affibody: Novel ultrasound contrast agents for targeted molecular ultrasound imaging of tumor. , 2015, Biomaterials.

[2]  Paul A. Dayton,et al.  Optical observation of lipid- and polymer-shelled ultrasound microbubble contrast agents , 2004 .

[3]  S. Wada,et al.  Collapse of a lipid-coated nanobubble and subsequent liposome formation , 2016, Scientific Reports.

[4]  W. Kühlbrandt,et al.  Cryo-EM enters a new era , 2014, eLife.

[5]  A. Exner,et al.  Ultrasound molecular imaging of ovarian cancer with CA-125 targeted nanobubble contrast agents. , 2017, Nanomedicine : nanotechnology, biology, and medicine.

[6]  A. Klibanov,et al.  Destruction of contrast agent microbubbles in the ultrasound field: the fate of the microbubble shell and the importance of the bubble gas content. , 2002, Academic radiology.

[7]  Sachin S Thakur,et al.  Stably engineered nanobubbles and ultrasound - An effective platform for enhanced macromolecular delivery to representative cells of the retina , 2017, PloS one.

[8]  L. Franken,et al.  Transmission Electron Microscopy as a Tool for the Characterization of Soft Materials: Application and Interpretation , 2017, Advanced science.

[9]  F. Kiessling,et al.  Physicochemical Characterization of the Shell Composition of PBCA-Based Polymeric Microbubbles. , 2017, Macromolecular bioscience.

[10]  Stuart D. Summerhayes.  Application and Interpretation , 2009 .

[11]  H. Stone,et al.  Interfacial Polygonal Nanopatterning of Stable Microbubbles , 2008, Science.

[12]  P. Lewin,et al.  Bursting Bubbles and Bilayers , 2012, Theranostics.

[13]  Stefaan C De Smedt,et al.  Design and evaluation of doxorubicin-containing microbubbles for ultrasound-triggered doxorubicin delivery: cytotoxicity and mechanisms involved. , 2010, Molecular therapy : the journal of the American Society of Gene Therapy.

[14]  M. Lattuada,et al.  Insertion of nanoparticle clusters into vesicle bilayers. , 2014, ACS nano.

[15]  Sriram Subramaniam,et al.  Electron tomography in nanoparticle imaging and analysis. , 2008, Nanomedicine.

[16]  N. Grigorieff,et al.  Direct visualisation of micelles of Pluronic block copolymers in aqueous solution by cryo-TEM , 1999 .

[17]  Johannes E. Schindelin,et al.  The ImageJ ecosystem: An open platform for biomedical image analysis , 2015, Molecular reproduction and development.

[18]  Luis Solorio,et al.  Nanobubble Ultrasound Contrast Agents for Enhanced Delivery of Thermal Sensitizer to Tumors Undergoing Radiofrequency Ablation , 2013, Pharmaceutical Research.

[19]  J. Ophir,et al.  IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control , 2008, IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control.

[20]  Douglas A Christensen,et al.  Drug-loaded nano/microbubbles for combining ultrasonography and targeted chemotherapy. , 2008, Ultrasonics.

[21]  P. Stewart,et al.  Polymer Structure and Conformation Alter the Antigenicity of Virus-like Particle-Polymer Conjugates. , 2017, Journal of the American Chemical Society.

[22]  K. Edwards,et al.  Cryo transmission electron microscopy of liposomes and related structures , 2000 .

[23]  Nico de Jong,et al.  High-speed optical observations of contrast agent destruction. , 2005, Ultrasound in medicine & biology.

[24]  Katherine W Ferrara,et al.  Ultrasound contrast microbubbles in imaging and therapy: physical principles and engineering , 2009, Physics in medicine and biology.

[25]  Luis Solorio,et al.  Acoustic characterization and pharmacokinetic analyses of new nanobubble ultrasound contrast agents. , 2013, Ultrasound in medicine & biology.

[26]  A. Arranja,et al.  Cytotoxicity and internalization of Pluronic micelles stabilized by core cross-linking. , 2014, Journal of controlled release : official journal of the Controlled Release Society.

[27]  Francois Vignon,et al.  Microbubble cavitation imaging , 2013, IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control.

[28]  P. Dayton,et al.  Mechanisms of contrast agent destruction , 2001, IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control.

[29]  P. Stewart.  Cryo-electron microscopy and cryo-electron tomography of nanoparticles. , 2017, Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology.

[30]  G. Winter,et al.  New doxorubicin-loaded phospholipid microbubbles for targeted tumor therapy: in-vivo characterization. , 2010, Journal of controlled release : official journal of the Controlled Release Society.

[31]  Luis Solorio,et al.  Formulation and characterization of echogenic lipid-Pluronic nanobubbles. , 2009, Molecular pharmaceutics.

[32]  Poojitha D. Yapa,et al.  Buoyant Velocity of Spherical and Nonspherical Bubbles/Droplets , 2000 .

[33]  A. Exner,et al.  Improving performance of nanoscale ultrasound contrast agents using N,N-diethylacrylamide stabilization. , 2017, Nanomedicine : nanotechnology, biology, and medicine.

[34]  P. Stewart,et al.  In Situ Characterization of Binary Mixed Polymer Brush-Grafted Silica Nanoparticles in Aqueous and Organic Solvents by Cryo-Electron Tomography. , 2015, Langmuir : the ACS journal of surfaces and colloids.

[35]  Thomas H. Epps,et al.  Structural characterization of amphiphilic homopolymer micelles using light scattering, SANS, and cryo-TEM. , 2013, Macromolecules.

[36]  Haoyan Zhou,et al.  Ultrasound imaging beyond the vasculature with new generation contrast agents. , 2015, Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology.