Gas Vesicle–Blood Interactions Enhance Ultrasound Imaging Contrast

Creators: Ling, Bill; Ko, Jeong Hoon; Stordy, Benjamin; Zhang, Yuwei; Didden, Tighe F.; Malounda, Dina; Swift, Margaret B.; Chan, Warren C. W.; Shapiro, Mikhail G.¹

1. California Institute of Technology

Abstract

Gas vesicles (GVs) are genetically encoded, air-filled protein nanostructures of broad interest for biomedical research and clinical applications, acting as imaging and therapeutic agents for ultrasound, magnetic resonance, and optical techniques. However, the biomedical applications of GVs as systemically injectable nanomaterials have been hindered by a lack of understanding of GVs' interactions with blood components, which can significantly impact in vivo behavior. Here, we investigate the dynamics of GVs in the bloodstream using a combination of ultrasound and optical imaging, surface functionalization, flow cytometry, and mass spectrometry. We find that erythrocytes and serum proteins bind to GVs and shape their acoustic response, circulation time, and immunogenicity. We show that by modifying the GV surface we can alter these interactions and thereby modify GVs' in vivo performance. These results provide critical insights for the development of GVs as agents for nanomedicine.

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Acknowledgement

The authors thank Prof. Bob Grubbs, Prof. Mark E. Davis, and Dr. Di Wu for helpful discussions; Dr. Hojin Kim for help with the aqueous SEC polymer characterization; Justin Lee for assistance with flow cytometry; Dr. Mona Shahgoli for assistance with small molecule mass spectrometry; the Caltech Cryo-EM Center for assistance with TEM; the Caltech CCE Multiuser Mass Spectrometry Lab for instrumentation to characterize small molecules and GVs; and Dr. Craig Simpson and Dr. Leanne Wynbenga-Groot, The Hospital for Sick Children, Toronto, Canada, for assistance with mass spectrometry for proteomic analysis. This research was supported by the National Institutes of Health (R01-EB018975 to M.G.S.) and the Rosen Bioengineering Center Pilot Grant. W.C.W.C. acknowledges the Canadian Institute of Health Research Grants FDN159932 and MOP-1301431, NMIN Network 2019-T3-01 and Canadian Research Chairs Program Grant 950-223824. J.H.K. was supported by the Kavli Nanoscience Institute Prize Postdoctoral Fellowship at the California Institute of Technology. B.L. was supported by the NIH/NRSA Pre-Doctoral Training Grant (T32GM07616) and the Caltech Center for Environmental and Microbial Interactions. B.S. thanks the Doctoral Completion Award. M.G.S. is a Howard Hughes Medical Institute Investigator.

Contributions

B.L. and J.H.K. contributed equally to this work. B.L, J.H.K., and M.G.S. conceptualized the research. B.L. performed the in vivo imaging experiments with assistance from M.B.S. J.H.K. designed polymer synthesis and gas vesicle functionalization reactions with assistance from T.F.D. J.H.K. and B.L. characterized the functionalized gas vesicles. B.L. performed the erythrocyte modeling and incubation experiments. B.S. and Y.Z. performed LC-MS/MS experiments and analyzed the data. D.M. prepared gas vesicles for experiments. All authors contributed to editing and revising the manuscript. M.G.S. and W.C.W.C. supervised the research.

Conflict of Interest

The authors declare no competing financial interest.

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