Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI

Autor: George J. Lu, Martin Kunth, Mikhail G. Shapiro, Audrey Lee-Gosselin, Anupama Lakshmanan, Arash Farhadi, David Maresca, Leif Schröder, Judy Yan, Christopher Witte, Raymond W. Bourdeau, Melissa Yin, Suchita P. Nety, Dina Malounda, F. Stuart Foster
Rok vydání: 2017
Předmět:
Zdroj: Nature Protocols. 12:2050-2080
ISSN: 1750-2799
1754-2189
DOI: 10.1038/nprot.2017.081
Popis: Gas vesicles are a unique class of gas-filled protein nanostructures whose physical properties allow them to serve as highly sensitive imaging agents for ultrasound and magnetic resonance imaging (MRI), detectable at sub-nanomolar concentrations. Here we provide a protocol for isolating gas vesicles from native and heterologous host organisms, functionalizing these nanostructures with moieties for targeting and fluorescence, characterizing their biophysical properties and imaging them using ultrasound and magnetic resonance imaging. Gas vesicles can be isolated from natural cyanobacterial and haloarchaeal host organisms or from E. coli expressing a heterologous gas vesicle gene cluster, and purified using buoyancy-assisted techniques. They can then be modified by replacing surface-bound proteins with engineered, heterologously expressed variants, or through chemical conjugation, resulting in altered mechanical, surface and targeting properties. Pressurized absorbance spectroscopy is used to characterize their mechanical properties, while dynamic light scattering and transmission electron microscopy are used to determine nanoparticle size and morphology, respectively. Gas vesicles can then be imaged with ultrasound in vitro and in vivo using pulse sequences optimized for their detection versus background. They can also be imaged with hyperpolarized xenon MRI using chemical exchange saturation transfer between gas vesicle-bound and dissolved xenon – a technique currently implemented in vitro. Taking 3–8 days to prepare, these genetically encodable nanostructures enable multi-modal, noninvasive biological imaging with high sensitivity and potential for molecular targeting.
Databáze: OpenAIRE
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