Viral nanoparticles as tools for intravital vascular imaging

Abstract

A significant impediment to the widespread use of noninvasive in vivo vascular imaging techniques is the current lack of suitable intravital imaging probes. We describe here a new strategy to use viral nanoparticles as a platform for the multivalent display of fluorescent dyes to image tissues deep inside living organisms. The bioavailable cowpea mosaic virus (CPMV) can be fluorescently labeled to high densities with no measurable quenching, resulting in exceptionally bright particles with in vivo dispersion properties that allow high-resolution intravital imaging of vascular endothelium for periods of at least 72 h. We show that CPMV nanoparticles can be used to visualize the vasculature and blood flow in living mouse and chick embryos to a depth of up to 500 μm. Furthermore, we show that the intravital visualization of human fibrosarcoma-mediated tumor angiogenesis using fluorescent CPMV provides a means to identify arterial and venous vessels and to monitor the neovascularization of the tumor microenvironment.

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Figure 1: Labeling of CPMV particles with fluorescent dyes.
Figure 2: Fluorescent dye–conjugated CPMV particles enable visualization of vasculature intravitally and in fixed tissues.
Figure 3: Intravital fluorescence imaging of chick CAM vasculature and subcellular localization of CPMV.
Figure 4: Comparison of intravital vascular staining intensity over time in the chick embryo.
Figure 5: Evaluation of tumor angiogenesis in an intravital CAM/HT1080 fibrosarcoma model.

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Acknowledgements

This study was supported by US National Institutes of Health grants R01 HL65738 and R21 HL72270 (to H.S.), AI47823, CA112075 and N01-CO-27181 (to M.M.), R01 CA55852 and R01 CA105412 (to J.Q.) and NSERC PDF-313420-2005 (to J.L.). The authors thank M. Wood (Scripps Microscopy Core Facility), X. Zhang, E. Rockenstein and N. MacLean (University of California San Diego) for their technical assistance.

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Correspondence to Marianne Manchester or Heidi Stuhlmann.

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A patent has been filed to cover the intellectual property described in this manuscript. This patent application has furthermore been exclusively licensed to a private third party.

Supplementary information

Supplementary Fig. 1

Cell adhesion and uptake is inhibited by coating of CPMV with PEG. (PDF 129 kb)

Supplementary Fig. 2

CPMV uptake is eliminated in chick embryos and reduced significantly in adult mice by PEG coating. (PDF 142 kb)

Supplementary Video 1

Fluorescent timelapse imaging of yolk sac of 11.5 d mouse embryo at 10 magnification 1 h after CPMV-A555 perfusion. (AVI 8067 kb)

Supplementary Video 2

Fluorescent timelapse imaging of chick embryo CAM vasculature at 20 magnification 30 min after CPMV-A555 perfusion. (AVI 3675 kb)

Supplementary Video 3

Fluorescent timelapse imaging of chick embryo CAM vasculature at 10 magnification 30 min after CPMV-A555 (left) and CPMV-PEG-FITC (right) coperfusion, duration; 11 s. (AVI 5813 kb)

Supplementary Video 4

Fluorescent timelapse imaging of internal vasculature of 11.5 d mouse embryo at 10 magnification 1 h after CPMV-A555 perfusion. (AVI 10674 kb)

Supplementary Methods (PDF 38 kb)

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Lewis, J., Destito, G., Zijlstra, A. et al. Viral nanoparticles as tools for intravital vascular imaging. Nat Med 12, 354–360 (2006). https://doi.org/10.1038/nm1368

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