Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles

Abstract

Viral nanoparticles are a novel class of biomolecular agents that take advantage of the natural circulatory and targeting properties of viruses to allow the development of therapeutics, vaccines and imaging tools. We have developed a multivalent nanoparticle platform based on the cowpea mosaic virus (CPMV) that facilitates particle labeling at high density with fluorescent dyes and other functional groups. Compared with other technologies, CPMV-based viral nanoparticles are particularly suited for long-term intravital vascular imaging because of their biocompatibility and retention in the endothelium with minimal side effects. The stable, long-term labeling of the endothelium allows the identification of vasculature undergoing active remodeling in real time. In this study, we describe the synthesis, purification and fluorescent labeling of CPMV nanoparticles, along with their use for imaging of vascular structure and for intravital vascular mapping in developmental and tumor angiogenesis models. Dye-labeled viral nanoparticles can be synthesized and purified in a single day, and imaging studies can be conducted over hours, days or weeks, depending on the application.

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Figure 1: The cowpea mosaic virus (CPMV) is a multivalent platform for fluorescent dye conjugation.
Figure 2: Preparation of shell-less avian embryos.
Figure 3: Microinjection of CPMV nanoparticles into mouse embryos.
Figure 4: Embryo imaging unit for intravital imaging of avian and mouse embryos.
Figure 5: Intravital vascular mapping of human tumors in the CAM.
Figure 6: Intravital imaging of mouse embryo vasculature.

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Acknowledgements

This study was supported by an American Heart Association Postdoctoral Fellowship (N.F.S.) as well as by the following grants: Canadian Institutes for Health Research grant MOP-84535 (J.D.L.), and US National Institutes of Health grants R01 CA112075 (M.M.), R01 HL 068648 (H.S.) and K99 EB009105 (N.F.S.).

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Authors

Contributions

H.S.L., A.A., J.D.L., A.Z. and H.S. developed the animal models and conducted intravital imaging experiments. N.F.S., G.D. and M.M. prepared and characterized the viral nanoparticles; H.S.L., N.F.S., A.A., H.S., M.M., A.Z. and J.D.L. wrote the paper.

Corresponding author

Correspondence to John D Lewis.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Movie 1: Real time imaging of murine embryo yolk sac vasculature.

CPMV-A555 was injected intravenously and widefield fluorescence images acquired every 300 msecs. Images shown reveal capillaries feeding into larger veins. (AVI 4300 kb)

Supplementary Movie 2: Real time imaging of murine embryo vasculature.

CPMV-A555 was injected intravenously and widefield fluorescence images acquired every 300 msecs. The top vessel represents an artery and the bottom vessel is a vein. (AVI 1601 kb)

Supplementary Movie 3: Real time imaging of blood cell transit within the avian embryo chorioallantoic membrane (CAM).

CPMV-A555 was injected intravenously and widefield fluorescence images acquired every 100 msecs. To the left is a vein and individual immune cells that have endocytosed labeled viral nanoparticles can be seen traversing across the CAM plexus (right of vein). (AVI 6257 kb)

Supplementary Movie 4: Real time imaging of pre-angiogenic tumor vasculature.

CPMV-A555 was injected intravenously and widefield fluorescence images acquired every 300 msecs. CPMV-A555 reveals the pre-angiogenic vascular network surrounding the primary tumor. Blood cell transit can be visualized within each vessel. (AVI 3822 kb)

Supplementary Movie 5: Real time imaging of tumor vasculature.

CPMV-A555 was injected intravenously and widefield fluorescence images acquired every 300 msecs. CPMV-A555 reveals a tortuous vascular network supporting the primary tumor (bottom middle). (AVI 4044 kb)

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Leong, H., Steinmetz, N., Ablack, A. et al. Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles. Nat Protoc 5, 1406–1417 (2010). https://doi.org/10.1038/nprot.2010.103

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