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:

Preparation of peptide-conjugated quantum dots for tumor vasculature-targeted imaging

Nature Protocols volume 3, pages 89–96 (2008)Cite this article

Abstract

To take full advantage of the unique optical properties of quantum dots (QDs) and expedite future near-infrared fluorescence (NIRF) imaging applications, QDs need to be effectively, specifically and reliably directed to a specific organ or disease site after systemic administration. Recently, we reported the use of peptide-conjugated QDs for non-invasive NIRF imaging of tumor vasculature markers in small animal models. In this protocol, we describe the detailed procedure for the preparation of such peptide-conjugated QDs using commercially available PEG-coated QDs and arginine-glycine-aspartic acid (RGD) peptides. Conjugation of the thiolated RGD peptide to the QDs was achieved through a heterobifunctional linker, 4-maleimidobutyric acid N-succinimidyl ester. Competitive cell binding assay, using 125I-echistatin as the radioligand, and live cell staining were carried out to confirm the successful attachment of the RGD peptides to the QD surface before in vivo imaging of tumor-bearing mice. In general, QD conjugation and in vitro validation of the peptide-conjugated QDs can be accomplished within 1–2 d; in vivo imaging will take another 1–2 d depending on the experimental design.

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: Synthesis of QD705-RGD.
Figure 2
Figure 3: Inhibition of 125I-echistatin (integrin αvβ3-specific) binding to αvβ3 integrin on U87MG cells by RGD peptide and QD705-RGD (n = 3, mean ± SD).
Figure 4: Staining of live human breast cancer MCF-7 cells (integrin αvβ3-negative) and human glioblastoma U87MG cells (integrin αvβ3-positive) using 1 nM of QD705-RGD.
Figure 5: In vivo imaging of U87MG tumor-bearing mice (yellow arrows) at 6 h post-injection of 200 pmol of QD705-RGD (left) or QD705 (right).

Similar content being viewed by others

References

  1. Cai, W., Hsu, A.R., Li, Z.B. & Chen, X. Are quantum dots ready for in vivo imaging in human subjects? Nanoscale Res. Lett. 2, 265–281 (2007).

    Article  CAS  Google Scholar 

  2. Michalet, X. et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307, 538–544 (2005).

    Article  CAS  Google Scholar 

  3. Medintz, I.L., Uyeda, H.T., Goldman, E.R. & Mattoussi, H. Quantum dot bioconjugates for imaging, labelling and sensing. Nat. Mater. 4, 435–446 (2005).

    Article  CAS  Google Scholar 

  4. Alivisatos, A.P., Gu, W. & Larabell, C. Quantum dots as cellular probes. Annu. Rev. Biomed. Eng. 7, 55–76 (2005).

    Article  CAS  Google Scholar 

  5. Li, Z.B., Cai, W. & Chen, X. Semiconductor quantum dots for in vivo imaging. J. Nanosci. Nanotechnol. 7, 2567–2581 (2007).

    Article  CAS  Google Scholar 

  6. Frangioni, J.V. In vivo near-infrared fluorescence imaging. Curr. Opin. Chem. Biol. 7, 626–634 (2003).

    Article  CAS  Google Scholar 

  7. Dubertret, B. et al. In vivo imaging of quantum dots encapsulated in phospholipid micelles. Science 298, 1759–1762 (2002).

    Article  CAS  Google Scholar 

  8. Voura, E.B., Jaiswal, J.K., Mattoussi, H. & Simon, S.M. Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy. Nat. Med. 10, 993–998 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Stroh, M. et al. Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo. Nat. Med. 11, 678–682 (2005).

    Article  CAS  Google Scholar 

  11. Kim, S. et al. Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping. Nat. Biotechnol. 22, 93–97 (2004).

    Article  CAS  Google Scholar 

  12. Zimmer, J.P. et al. Size series of small indium arsenide-zinc selenide core-shell nanocrystals and their application to in vivo imaging. J. Am. Chem. Soc. 128, 2526–2527 (2006).

    Article  CAS  Google Scholar 

  13. Thorne, R.G. & Nicholson, C. In vivo diffusion analysis with quantum dots and dextrans predicts the width of brain extracellular space. Proc. Natl. Acad. Sci. USA 103, 5567–5572 (2006).

    Article  CAS  Google Scholar 

  14. Mammen, M., Chio, S. & Whitesides, G.M. Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew. Chem. Int. Ed. Engl. 37, 2755–2794 (1998).

    Article  CAS  Google Scholar 

  15. Cai, W. & Chen, X. Anti-angiogenic cancer therapy based on integrin αvβ3 antagonism. Anti-Cancer Agents Med. Chem. 6, 407–428 (2006).

    Article  CAS  Google Scholar 

  16. Cai, W., Rao, J., Gambhir, S.S. & Chen, X. How molecular imaging is speeding up anti-angiogenic drug development. Mol. Cancer Ther. 5, 2624–2633 (2006).

    Article  CAS  Google Scholar 

  17. Bergers, G. & Benjamin, L.E. Tumorigenesis and the angiogenic switch. Nat. Rev. Cancer 3, 401–410 (2003).

    Article  CAS  Google Scholar 

  18. Hood, J.D. & Cheresh, D.A. Role of integrins in cell invasion and migration. Nat. Rev. Cancer 2, 91–100 (2002).

    Article  Google Scholar 

  19. Hynes, R.O. Integrins: bidirectional, allosteric signaling machines. Cell 110, 673–687 (2002).

    Article  CAS  Google Scholar 

  20. Cai, W. et al. Peptide-labeled near-infrared quantum dots for imaging tumor vasculature in living subjects. Nano Lett. 6, 669–676 (2006).

    Article  CAS  Google Scholar 

  21. Hermanson, G.T. Bioconjugate Techniques (Academic Press, San Diego, CA, USA, 1996).

    Google Scholar 

  22. Cai, W. et al. In vitro and in vivo characterization of 64Cu-labeled Abegrin, a humanized monoclonal antibody against integrin αvβ3 . Cancer Res. 66, 9673–9681 (2006).

    Article  CAS  Google Scholar 

  23. Liu, Z. et al. In vivo biodistribution and highly efficient tumour targeting of carbon nanotubes in mice. Nat. Nanotechnol. 2, 47–52 (2007).

    Article  CAS  Google Scholar 

  24. Cao, Q. et al. Combination of integrin siRNA and irradiation for breast cancer therapy. Biochem. Biophys. Res. Commun. 351, 726–732 (2006).

    Article  CAS  Google Scholar 

  25. Pradhan, N., Battaglia, D.M., Liu, Y. & Peng, X. Efficient, stable, small, and water-soluble doped ZnSe nanocrystal emitters as non-cadmium biomedical labels. Nano Lett. 7, 312–317 (2007).

    Article  CAS  Google Scholar 

  26. Pradhan, N. & Peng, X. Efficient and color-tunable Mn-doped ZnSe nanocrystal emitters: control of optical performance via greener synthetic chemistry. J. Am. Chem. Soc. 129, 3339–3347 (2007).

    Article  CAS  Google Scholar 

  27. So, M.K., Xu, C., Loening, A.M., Gambhir, S.S. & Rao, J. Self-illuminating quantum dot conjugates for in vivo imaging. Nat. Biotechnol. 24, 339–343 (2006).

    Article  CAS  Google Scholar 

  28. So, M.K., Loening, A.M., Gambhir, S.S. & Rao, J. Creating self-illuminating quantum dot conjugates. Nat. Protoc. 1, 1160–1164 (2006).

    Article  CAS  Google Scholar 

  29. Mulder, W.J. et al. Quantum dots with a paramagnetic coating as a bimodal molecular imaging probe. Nano Lett. 6, 1–6 (2006).

    Article  CAS  Google Scholar 

  30. Cai, W., Chen, K., Li, Z.B., Gambhir, S.S. & Chen, X. Dual-function probe for PET and near-infrared fluorescence imaging of tumor vasculature. J. Nucl. Med. 48, 1862–1870 (2007).

    Article  CAS  Google Scholar 

  31. Cai, W. et al. Quantitative PET of EGFR expression in xenograft-bearing mice using 64Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody. Eur. J. Nucl. Med. Mol Imaging. 34, 850–858 (2007).

    Article  CAS  Google Scholar 

  32. Cai, W. et al. PET of vascular endothelial growth factor receptor expression. J. Nucl. Med. 47, 2048–2056 (2006).

    CAS  PubMed  Google Scholar 

  33. Levenson, R.M. Spectral imaging and pathology: seeing more. Lab. Med. 35, 244–251 (2004).

    Article  Google Scholar 

  34. Mansfield, J.R., Gossage, K.W., Hoyt, C.C. & Levenson, R.M. Autofluorescence removal, multiplexing, and automated analysis methods for in-vivo fluorescence imaging. J. Biomed. Opt. 10, 41207 (2005).

    Article  Google Scholar 

  35. Xiong, J.P. et al. Crystal structure of the extracellular segment of integrin αvβ3 . Science 294, 339–345 (2001).

    Article  CAS  Google Scholar 

  36. Xiong, J.P. et al. Crystal structure of the extracellular segment of integrin αvβ3 in complex with an Arg-Gly-Asp ligand. Science 296, 151–155 (2002).

    Article  CAS  Google Scholar 

  37. Cai, W., Zhang, X., Wu, Y. & Chen, X. A thiol-reactive 18F-labeling agent, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM), and the synthesis of RGD peptide-based tracer for PET imaging of αvβ3 integrin expression. J. Nucl. Med. 47, 1172–1180 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Jue, R., Lambert, J.M., Pierce, L.R. & Traut, R.R. Addition of sulfhydryl groups to Escherichia coli ribosomes by protein modification with 2-iminothiolane (methyl 4-mercaptobutyrimidate). Biochemistry 17, 5399–5406 (1978).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Cancer Institute (NCI) Center of Cancer Nanotechnology Excellence (CCNE) grant U54CA119367 and R21 CA121842.

Author information

Authors and Affiliations

  1. Department of Radiology and Bio-X Program, Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, Stanford, 94305, California, USA

    Weibo Cai & Xiaoyuan Chen

Authors
  1. Weibo Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaoyuan Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

W.C. and X.C. did the conjugation. W.C. did the imaging experiments and analyzed the data. W.C. and X.C. designed and carried out research. W.C. and X.C. wrote the manuscript.

Corresponding author

Correspondence to Xiaoyuan Chen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cai, W., Chen, X. Preparation of peptide-conjugated quantum dots for tumor vasculature-targeted imaging. Nat Protoc 3, 89–96 (2008). https://doi.org/10.1038/nprot.2007.478

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.478

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

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing