Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles

Nature Protocols volume 5, pages 1406–1417 (2010)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Carmeliet, P. Angiogenesis in life, disease and medicine. Nature 438, 932–936 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Destito, G., Schneemann, A. & Manchester, M. Biomedical nanotechnology using virus-based nanoparticles. Curr. Top Microbiol. Immunol. 327, 95–122 (2009).

    CAS  PubMed  Google Scholar 

  3. Manchester, M. & Singh, P. Virus-based nanoparticles (VNPs): platform technologies for diagnostic imaging. Adv. Drug Deliv. Rev. 58, 1505–1522 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Young, M., Willits, D., Uchida, M. & Douglas, T. Plant viruses as biotemplates for materials and their use in nanotechnology. Annu. Rev. Phytopathol. 46, 361–384 (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Lewis, J.D. et al. Viral nanoparticles as tools for intravital vascular imaging. Nat. Med. 12, 354–360 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Lin, T. et al. The refined crystal structure of cowpea mosaic virus at 2.8 A resolution. Virology 265, 20–34 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Chatterji, A. et al. New addresses on an addressable virus nanoblock; uniquely reactive Lys residues on cowpea mosaic virus. Chem. Biol. 11, 855–863 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Wang, Q., Kaltgrad, E., Lin, T., Johnson, J.E. & Finn, M.G. Natural supramolecular building blocks. Wild-type cowpea mosaic virus. Chem. Biol. 9, 805–811 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Medintz, I.L. et al. Decoration of discretely immobilized cowpea mosaic virus with luminescent quantum dots. Langmuir 21, 5501–5510 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Singh, P. et al. Bio-distribution, toxicity and pathology of cowpea mosaic virus nanoparticles in vivo . J. Control Release. 120, 41–50 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sapsford, K.E. et al. A cowpea mosaic virus nanoscaffold for multiplexed antibody conjugation: application as an immunoassay tracer. Biosens. Bioelectron. 21, 1668–1673 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Chatterji, A. et al. Chemical conjugation of heterologous proteins on the surface of Cowpea mosaic virus. Bioconjug. Chem. 15, 807–813 (2004).

    Article  CAS  PubMed  Google Scholar 

  13. Sen Gupta, S. et al. Accelerated bioorthogonal conjugation: a practical method for the ligation of diverse functional molecules to a polyvalent virus scaffold. Bioconjug. Chem. 16, 1572–1579 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Martin, B.D. et al. An engineered virus as a bright fluorescent tag and scaffold for cargo proteins—capture and transport by gliding microtubules. J. Nanosci. Nanotechnol. 6, 2451–2460 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Brunel, F.M. et al. Hydrazone ligation strategy to assemble multifunctional viral nanoparticles for cell imaging and tumor targeting. Nano. Lett. 10, 1093–1097 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Destito, G., Yeh, R., Rae, C.S., Finn, M.G. & Manchester, M. Folic acid-mediated targeting of cowpea mosaic virus particles to tumor cells. Chem. Biol. 14, 1152–1162 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Raja, K.S. et al. Hybrid virus-polymer materials. 1. Synthesis and properties of PEG-decorated cowpea mosaic virus. Biomacromolecules 4, 472–476 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Steinmetz, N.F. & Manchester, M. PEGylated viral nanoparticles for biomedicine: the impact of PEG chain length on VNP cell interactions in vitro and ex vivo . Biomacromolecules 10, 784–792 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gonzalez, M.J., Plummer, E.M., Rae, C.S. & Manchester, M. Interaction of Cowpea mosaic virus (CPMV) nanoparticles with antigen presenting cells in vitro and in vivo . PLoS One 4, e7981 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Shriver, L.P., Koudelka, K.J. & Manchester, M. Viral nanoparticles associate with regions of inflammation and blood brain barrier disruption during CNS infection. J. Neuroimmunol. 211, 66–72 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Brennan, F.R., Jones, T.D. & Hamilton, W.D. Cowpea mosaic virus as a vaccine carrier of heterologous antigens. Mol. Biotechnol. 17, 15–26 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Lomonossoff, G.P. & Shanks, M. The nucleotide sequence of cowpea mosaic virus B RNA. EMBO J. 2, 2253–2258 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Nicholas, B.L. et al. Characterization of the immune response to canine parvovirus induced by vaccination with chimaeric plant viruses. Vaccine 20, 2727–2734 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Yang, C.S. et al. Nanoparticle-based in vivo investigation on blood-brain barrier permeability following ischemia and reperfusion. Anal. Chem. 76, 4465–4471 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Hainfeld, J.F., Slatkin, D.N., Focella, T.M. & Smilowitz, H.M. Gold nanoparticles: a new X-ray contrast agent. Br. J. Radiol. 79, 248–253 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Josephson, L., Kircher, M.F., Mahmood, U., Tang, Y. & Weissleder, R. Near-infrared fluorescent nanoparticles as combined MR/optical imaging probes. Bioconjug. Chem. 13, 554–560 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Kim, D., Park, S., Lee, J.H., Jeong, Y.Y. & Jon, S. Antibiofouling polymer-coated gold nanoparticles as a contrast agent for in vivo X-ray computed tomography imaging. J. Am. Chem. Soc. 129, 7661–7665 (2007).

    Article  CAS  PubMed  Google Scholar 

  28. Lee, P.J. & Peyman, G.A. Visualization of the retinal and choroidal microvasculature by fluorescent liposomes. Methods Enzymol. 373, 214–233 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Zheng, J., Liu, J., Dunne, M., Jaffray, D.A. & Allen, C. In vivo performance of a liposomal vascular contrast agent for CT and MR-based image guidance applications. Pharm. Res. 24, 1193–1201 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. Rizzo, V., Steinfeld, R., Kyriakides, C. & DeFouw, D.O. The microvascular unit of the 6-day chick chorioallantoic membrane: a fluorescent confocal microscopic and ultrastructural morphometric analysis of endothelial permselectivity. Microvasc. Res. 46, 320–332 (1993).

    Article  CAS  PubMed  Google Scholar 

  31. Jilani, S.M. et al. Selective binding of lectins to embryonic chicken vasculature. J. Histochem. Cytochem. 51, 597–604 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Pardanaud, L., Altmann, C., Kitos, P., Dieterlen-Lievre, F. & Buck, C.A. Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells. Development 100, 339–349 (1987).

    CAS  PubMed  Google Scholar 

  33. Jayagopal, A., Russ, P.K. & Haselton, F.R. Surface engineering of quantum dots for in vivo vascular imaging. Bioconjug. Chem. 18, 1424–1433 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Larson, D.R. et al. Water-soluble quantum dots for multiphoton fluorescence imaging in vivo . Science 300, 1434–1436 (2003).

    Article  CAS  PubMed  Google Scholar 

  35. Khurana, M., Moriyama, E.H., Mariampillai, A. & Wilson, B.C. Intravital high-resolution optical imaging of individual vessel response to photodynamic treatment. J. Biomed. Opt. 13, 040502 (2008).

    Article  PubMed  Google Scholar 

  36. Mitra, S. & Foster, T.H. In vivo confocal fluorescence imaging of the intratumor distribution of the photosensitizer mono-L-aspartylchlorin-e6. Neoplasia 10, 429–438 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Bogen, S., Pak, J., Garifallou, M., Deng, X. & Muller, W.A. Monoclonal antibody to murine PECAM-1 (CD31) blocks acute inflammation in vivo . J. Exp. Med. 179, 1059–1064 (1994).

    Article  CAS  PubMed  Google Scholar 

  38. Chan, W.H., Shiao, N.H. & Lu, P.Z. CdSe quantum dots induce apoptosis in human neuroblastoma cells via mitochondrial-dependent pathways and inhibition of survival signals. Toxicol. Lett. 167, 191–200 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Cho, S.J. et al. Long-term exposure to CdTe quantum dots causes functional impairments in live cells. Langmuir 23, 1974–1980 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Hardman, R. A toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors. Environ. Health Perspect. 114, 165–172 (2006).

    Article  PubMed  Google Scholar 

  41. Prato, M., Kostarelos, K. & Bianco, A. Functionalized carbon nanotubes in drug design and discovery. Acc. Chem. Res. 41, 60–68 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Takagi, A. et al. Induction of mesothelioma in p53+/− mouse by intraperitoneal application of multi-wall carbon nanotube. J. Toxicol. Sci. 33, 105–116 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. Lasagna-Reeves, C. et al. Bioaccumulation and toxicity of gold nanoparticles after repeated administration in mice. Biochem. Biophys. Res. Commun. 393, 649–655.

    Article  CAS  PubMed  Google Scholar 

  44. Ochoa, W.F., Chatterji, A., Lin, T. & Johnson, J.E. Generation and structural analysis of reactive empty particles derived from an icosahedral virus. Chem. Biol. 13, 771–778 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Rae, C. et al. Chemical addressability of ultraviolet-inactivated viral nanoparticles (VNPs). PLoS One 3, e3315 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Saunders, K., Sainsbury, F. & Lomonossoff, G.P. Efficient generation of cowpea mosaic virus empty virus-like particles by the proteolytic processing of precursors in insect cells and plants. Virology 393, 329–337 (2009).

    Article  CAS  PubMed  Google Scholar 

  47. Liebermann, H. & Mentel, R. Quantification of adenovirus particles. J. Virol. Methods. 50, 281–291 (1994).

    Article  CAS  PubMed  Google Scholar 

  48. Wang, F. In Vitro Cellular & Developmental Biology—Animal (Springer Berlin, Heidelberg, Germany, 2003).

  49. Zijlstra, A., Lewis, J., Degryse, B., Stuhlmann, H. & Quigley, J.P. The inhibition of tumor cell intravasation and subsequent metastasis via regulation of in vivo tumor cell motility by the tetraspanin CD151. Cancer Cell 13, 221–234 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Carrillo-Tripp, M. et al. VIPERdb2: an enhanced and web API enabled relational database for structural virology. Nucleic Acids Res. 37, D436–D442 (2009).

    Article  CAS  PubMed  Google Scholar 

Download references

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.).

Author information

Authors and Affiliations

  1. Translational Prostate Cancer Research Group, London Regional Cancer Program, London, Ontario, Canada

    Hon Sing Leong, Amber Ablack & John D Lewis

  2. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, USA

    Nicole F Steinmetz & Marianne Manchester

  3. Department of Molecular Biology and Center for Integrative Molecular Biosciences, The Scripps Research Institute, La Jolla, California, USA

    Nicole F Steinmetz

  4. Kyowa Hakko Kirin California, La Jolla, California, USA

    Giuseppe Destito

  5. Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Andries Zijlstra

  6. Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, USA

    Heidi Stuhlmann

  7. Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada

    John D Lewis

Authors
  1. Hon Sing Leong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicole F Steinmetz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Amber Ablack
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Giuseppe Destito
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andries Zijlstra
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Heidi Stuhlmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Marianne Manchester
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. John D Lewis
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2010.103

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer