Multiplexed 129Xe HyperCEST MRI Detection of Genetically Reconstituted Bacterial Protein Nanoparticles in Human Cancer Cells.

Ryota Mizushima,Atsuko H. Iwane,Atsuomi Kimura,Hideaki Fujiwara,Tomonobu M. Watanabe,Kanako Inoue

doi:10.1155/2020/5425934

Abstract

Gas vesicle nanoparticles (GVs) are gas-containing protein assemblies expressed in bacteria and archaea. Recently, GVs have gained considerable attention for biotechnological applications as genetically encodable contrast agents for MRI and ultrasonography. However, at present, the practical use of GVs is hampered by a lack of robust methodology for their induction into mammalian cells. Here, we demonstrate the genetic reconstitution of protein nanoparticles with characteristic bicone structures similar to natural GVs in a human breast cancer cell line KPL-4 and genetic control of their size and shape through expression of reduced sets of humanized gas vesicle genes cloned into Tol2 transposon vectors, referencing the natural gas vesicle gene clusters of the cyanobacteria planktothrix rubescens/agardhii. We then report the utility of these nanoparticles as multiplexed, sensitive, and genetically encoded contrast agents for hyperpolarized xenon chemical exchange saturation transfer (HyperCEST) MRI.

Highlights

Gas vesicle nanoparticles (GVs) are gas-containing spindle(or bicone-) shaped protein nanostructures with dimensions ranging from tens to hundreds of nm that are expressed in the cyanobacteria, algae, and Gram-positive bacteria
E genes used in this study are listed in Table 1. ese praGV genes with codons optimized for expression in mammalian hosts were synthesized and cloned into Tol2 transposon vectors [15, 16, 20] under the control of tetracycline-inducible elements (Tet-On) [17]
We showed that expression of reduced GV gene sets derived from Planktothrix rubescens/agardhii in mammalian cells resulted in the formation of GV-like particles (GVLPs) in the cell and they could be functionalized as a genetically encoded HyperCEST MRI contrast agents

Summary

Introduction

Gas vesicle nanoparticles (GVs) are gas-containing spindle(or bicone-) shaped protein nanostructures with dimensions ranging from tens to hundreds of nm that are expressed in the cyanobacteria, algae, and Gram-positive bacteria. While GVs have been studied in the field of microbiology for several decades, they have gained significant attention in recent years for their potential use for antigenic peptide display in vaccination [4] and as genetically encodable contrast agents for ultrasonography [5,6,7] and HyperCEST MRI [8, 9] Among these applications, HyperCEST MRI, which utilizes laser-polarized xenon-129 (HPXe) and chemical exchange saturation transfer (CEST) [10] to yield unprecedented enhanced MRI detection sensitivity, is of particular interest as GVs hold the potential to enable functional molecular imaging that is unfeasible in conventional thermally polarized proton MRI. (In principle, GV concentrations of as low as pM–nM can be detected) In this context, GVs can be thought of as an MRI analog of the green fluorescent protein (GFP) optical imaging reporter, with a genetically encodable nature and multiplexing capability facilitated by ready modulation of their size and shape, similar to the multicolor variant of GFPs [8]. Despite their attractive features for imaging cellular/molecular processes, the practical in vivo use of GVs as genetically encoded contrast agents is at present hampered by a lack of robust techniques to introduce GVs into

Methods

Results

Conclusion