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

Nature Protocols volume 12, pages 2050–2080 (2017)Cite this article

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Abstract

Gas vesicles (GVs) are a unique class of gas-filled protein nanostructures that are detectable at subnanomolar concentrations and whose physical properties allow them to serve as highly sensitive imaging agents for ultrasound and MRI. Here we provide a protocol for isolating GVs 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 MRI. GVs can be isolated from natural cyanobacterial and haloarchaeal host organisms or from Escherichia coli expressing a heterologous GV 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, whereas dynamic light scattering (DLS)and transmission electron microscopy (TEM) are used to determine nanoparticle size and morphology, respectively. GVs 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 GV-bound and dissolved xenon—a technique currently implemented in vitro. Taking 3–8 d to prepare, these genetically encodable nanostructures enable multimodal, noninvasive biological imaging with high sensitivity and potential for molecular targeting.

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Figure 1: Flowchart illustrating the experimental design and workflow for gas vesicle (GV) production, quantification, characterization, functionalization and imaging applications (step numbers are given in yellow circles).
Figure 2: Equipment setup and expected results for native production and purification of GVs.
Figure 3: Collapsometry setup.
Figure 4: Ultrasound setup for in vitro and in vivo imaging.
Figure 5: Required elements for assembly of the gas-delivery manifold for 129Xe-MRI.
Figure 6: 129Xe HyperCEST MRI.
Figure 7: Anticipated results for GV characterization and in vitro ultrasound imaging.
Figure 8: Anticipated results for in vitro and in vivo ultrasound imaging.

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References

  1. Ferrara, K., Pollard, R. & Borden, M. Ultrasound microbubble contrast agents: fundamentals and application to gene and drug delivery. Annu. Rev. Biomed. Eng. 9, 415–447 (2007).

    Article  CAS  Google Scholar 

  2. Kaufmann, B.A. & Lindner, J.R. Molecular imaging with targeted contrast ultrasound. Curr. Opin. Biotechnol. 18, 11–16 (2007).

    Article  CAS  Google Scholar 

  3. Weissleder, R. et al. Ultrasmall superparamagnetic iron oxide: characterization of a new class of contrast agents for MR imaging. Radiology 175, 489–493 (1990).

    Article  CAS  Google Scholar 

  4. Lee, J.-H. et al. Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imaging. Nat. Med. 13, 95–99 (2007).

    Article  CAS  Google Scholar 

  5. Caravan, P., Ellison, J.J., McMurry, T.J. & Lauffer, R.B. Gadolinium (III) chelates as MRI contrast agents: structure, dynamics, and applications. Chem. Rev. 99, 2293–2352 (1999).

    Article  CAS  Google Scholar 

  6. Walsby, A.E. Gas vesicles. Microbiol. Rev. 58, 94–144 (1994).

    Article  CAS  Google Scholar 

  7. Pfeifer, F. Distribution, formation and regulation of gas vesicles. Nat. Rev. Microbiol. 10, 705–715 (2012).

    Article  CAS  Google Scholar 

  8. Shapiro, M.G. et al. Biogenic gas nanostructures as ultrasonic molecular reporters. Nat. Nanotechnol. 9, 311–316 (2014).

    Article  CAS  Google Scholar 

  9. Maresca, D. et al. Nonlinear ultrasound imaging of nanoscale acoustic biomolecules. Appl. Phys. Lett. 110, 073704 (2017).

    Article  Google Scholar 

  10. Cherin, E. et al. Acoustic behavior of Halobacterium salinarum gas vesicles in the high-frequency range: experiments and modeling. Ultrasound Med. Biol. 43, 1016–1030 (2017).

    Article  Google Scholar 

  11. Shapiro, M.G. et al. Genetically encoded reporters for hyperpolarized xenon magnetic resonance imaging. Nat. Chem. 6, 629–634 (2014).

    Article  CAS  Google Scholar 

  12. Hane, F.T. et al. In vivo detection of cucurbit[6]uril, a hyperpolarized xenon contrast agent for a xenon magnetic resonance imaging biosensor. Sci. Rep. 7, 41027 (2017).

    Article  CAS  Google Scholar 

  13. Barskiy, D.A. et al. NMR hyperpolarization techniques of gases. Chem. Eur. J. 23, 725–751 (2017).

    Article  CAS  Google Scholar 

  14. Lakshmanan, A. et al. Molecular engineering of acoustic protein nanostructures. ACS Nano 10, 7314–7322 (2016).

    Article  CAS  Google Scholar 

  15. Li, N. & Cannon, M.C. Gas vesicle genes identified in Bacillus megaterium and functional expression in Escherichia coli. J. Bacteriol. 180, 2450–2458 (1998).

    Article  CAS  Google Scholar 

  16. Gilad, A.A. & Shapiro, M.G. Molecular imaging in synthetic biology, and synthetic biology in molecular imaging. Mol. Imaging Biol. 19, 373–378 (2017).

    Article  CAS  Google Scholar 

  17. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. PNAS 109, E690–E697 (2012).

    Article  CAS  Google Scholar 

  18. Schröder, L. Chapter 8 HyperCEST Imaging. In Chemical Exchange Saturation Transfer Imaging: Advances and Applications. pp 121–158 (Pan Stanford Publishing, 2017).

    Google Scholar 

  19. Schröder, L., Chapter 17 - Xenon Biosensor HyperCEST MRI A2 - Albert, Mitchell S. In Hyperpolarized xenon for NMR and MRI (ed. Hane, F.T.) pp 263–277 (Academic Press, Boston, 2017).

  20. Witte, C. et al. Hyperpolarized xenon for NMR and MRI applications. JoVE 67, E4268 (2012).

    Google Scholar 

  21. Witte, C., Kunth, M., Rossella, F. & Schröder, L. Observing and preventing rubidium runaway in a direct-infusion xenon-spin hyperpolarizer optimized for high-resolution hyper-CEST (chemical exchange saturation transfer using hyperpolarized nuclei) NMR. J. Chem. Phys. 140, 084203 (2014).

    Article  CAS  Google Scholar 

  22. Nikolaou, P. et al. Near-unity nuclear polarization with an open-source 129Xe hyperpolarizer for NMR and MRI. PNAS 110, 14150–14155 (2013).

    Article  CAS  Google Scholar 

  23. Kunth, M., Witte, C., Hennig, A. & Schröder, L. Identification, classification, and signal amplification capabilities of high-turnover gas binding hosts in ultra-sensitive NMR. Chem. Sci. 6, 6069–6075 (2015).

    Article  CAS  Google Scholar 

  24. Kunth, M., Witte, C. & Schröder, L. Continuous-wave saturation considerations for efficient xenon depolarization. NMR Biomed. 28, 601–606 (2015).

    Article  CAS  Google Scholar 

  25. Smith, R. & Peat, A. Growth and gas-vacuole development in vegetative cells of Anabaena flos-aquae. Arch. Microbiol. 58, 117–126 (1967).

    Google Scholar 

  26. Buchholz, B., Hayes, P. & Walsby, A. The distribution of the outer gas vesicle protein, GvpC, on the Anabaena gas vesicle, and its ratio to GvpA. Microbiology 139, 2353–2363 (1993).

    CAS  Google Scholar 

  27. Simon, R.D. Morphology and protein composition of gas vesicles from wild type and gas vacuole defective strains of Halobacterium salinarium strain 5. Microbiology 125, 103–111 (1981).

    Article  CAS  Google Scholar 

  28. Kunth, M., Witte, C. & Schröder, L. Quantitative chemical exchange saturation transfer with hyperpolarized nuclei (qHyper-CEST): sensing xenon-host exchange dynamics and binding affinities by NMR. J. Chem. Phys. 141, 194202 (2014).

    Article  CAS  Google Scholar 

  29. Zaiss, M., Schnurr, M. & Bachert, P. Analytical solution for the depolarization of hyperpolarized nuclei by chemical exchange saturation transfer between free and encapsulated xenon (HyperCEST). J. Chem. Phys. 136, 144106 (2012).

    Article  Google Scholar 

Download references

Acknowledgements

This research was supported by the National Institutes of Health (R01-EB018975 to M.G.S.), DARPA (W911NF-14-1-0111 to M.G.S.), CIHR (grant no. FDN148367 to F.S.F.) and HFSP (RGP0050/2016 to M.G.S. and L.S.). A.L. is supported by an NSF graduate research fellowship (no. 1144469). A.F. is supported by an NSERC graduate fellowship. S.P.N. is supported by a Caltech Summer Undergraduate Research Fellowship. D. Maresca is supported by an HFSP Cross-Disciplinary Postdoctoral Fellowship. Research in the Shapiro laboratory is also supported by the Heritage Medical Research Institute, the Burroughs Wellcome Fund, the Pew Charitable Trust, the Sontag Foundation and the David and Lucile Packard Foundation. Research in the Schröder laboratory is also supported by the Michael J. Fox Foundation for Parkinson's Research (no. 12549) and a Koselleck Grant by the German Research Foundation (DFG; project SCHR 995/5-1). We acknowledge the Jensen Lab and the Beckman Resource Center for Electron Microscopy (EM) at the California Institute of Technology for technical guidance and for allowing us to use their resources. The EM facility is funded by the Beckman Foundation, the Gordon and Betty Moore Foundation, the Agouron Institute and the HHMI.

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Author notes

  1. Anupama Lakshmanan, George J Lu, Arash Farhadi and Suchita P Nety: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA

    Anupama Lakshmanan & Arash Farhadi

  2. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA

    George J Lu, Suchita P Nety, Audrey Lee-Gosselin, David Maresca, Raymond W Bourdeau, Dina Malounda & Mikhail G Shapiro

  3. Department of Structural Biology, Molecular Imaging, Leibniz-Forschungsinstitute für Molekulare Pharmakologie, Berlin, Germany

    Martin Kunth, Christopher Witte & Leif Schröder

  4. Sunnybrook Research Institute, University of Toronto, Toronto, Ontario, Canada

    Melissa Yin, Judy Yan & F Stuart Foster

  5. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

    F Stuart Foster

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  1. Anupama Lakshmanan
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Contributions

A.L., G.J.L., A.F., S.P.N. and M.G.S. conceived the manuscript layout and coordinated its writing. A.L., G.J.L., A.F., S.P.N., A.L.-G., D. Maresca, D. Malounda, R.W.B. and M.G.S developed methods for GV production, functionalization, and characterization, as well as for ultrasound imaging. M.Y., J.Y. and F.S.F. contributed methods for in vivo ultrasound imaging of GVs. M.K., C.W., L.S. and M.G.S. contributed methods for hyperpolarized xenon MRI of GVs. All authors contributed to writing the manuscript. A.L. compiled and edited the protocol and G.J.L. assembled the figures.

Corresponding author

Correspondence to Mikhail G Shapiro.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Ultrasound imaging of Mega GVs

B-mode ultrasound images of purified, wild-type Ana GVs (OD500,ps: 2.2) versus a purified and unclustered batch of Mega GVs at (a) equal molarity, (b) equal protein concentration and (c) equal gas fraction. Scale bars are 1 mm. Images were acquired using the Verasonics L22-14v transducer and the ray-lines script with the following parameters: transmit frequency: 18MHz, number of cycles of the transmitted pulse: 6, F number: 2, imaging voltage: 3V, with the transducer focus (8 mm depth) aligned close to the center of the sample well. Images were processed and analyzed using MATLAB. Images are shown before (top panel) and after collapse (bottom panel) using a high-power burst from the transducer at 25V for 10 s. (d) Quantification of ultrasound signal was performed by selecting a region of interest (ROI) of defined size within the sample well and calculating the mean intensity per pixel for the selected ROI, after post-collapse background subtraction (n=12 for Ana GVs, n=4 for Mega GVs at each condition shown in a, b and c; error bars are SEM).

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Supplementary Figure 1. (PDF 306 kb)

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Lakshmanan, A., Lu, G., Farhadi, A. et al. Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI. Nat Protoc 12, 2050–2080 (2017). https://doi.org/10.1038/nprot.2017.081

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