Bioorthogonal chemistry amplifies nanoparticle binding and enhances the sensitivity of cell detection

Journal name:
Nature Nanotechnology
Volume:
5,
Pages:
660–665
Year published:
DOI:
doi:10.1038/nnano.2010.148
Received
Accepted
Published online

Abstract

Nanoparticles have emerged as key materials for biomedical applications because of their unique and tunable physical properties, multivalent targeting capability, and high cargo capacity1, 2. Motivated by these properties and by current clinical needs, numerous diagnostic3, 4, 5, 6, 7, 8, 9, 10 and therapeutic11, 12, 13 nanomaterials have recently emerged. Here we describe a novel nanoparticle targeting platform that uses a rapid, catalyst-free cycloaddition as the coupling mechanism. Antibodies against biomarkers of interest were modified with trans-cyclooctene and used as scaffolds to couple tetrazine-modified nanoparticles onto live cells. We show that the technique is fast, chemoselective, adaptable to metal nanomaterials, and scalable for biomedical use. This method also supports amplification of biomarker signals, making it superior to alternative targeting techniques including avidin/biotin.

At a glance

Figures

  1. Overview of BOND.
    Figure 1: Overview of BOND.

    a, Schematic showing the conjugation chemistry between antibody and nanoparticle. The diagram is a schematic and not to scale. b, Comparative sizes (to scale) of a representative mouse IgG2a antibody (lysine residues available for TCO modification via amine-reactive chemistry are shown in yellow), a TCO modification and an avidin protein for comparison. Tetrazine is similar in size to TCO (~200 Da). Protein structures and sizes were obtained from the Protein Data Bank (antibody, 1IGT; avidin, 3FDC) c, Application of BOND for one-step (direct) and two-step targeting of nanoparticles to cells. Note that the antibody and tetrazine are present in multiple copies per nanoparticle (~2–3 antibodies, Ab; 84 tetrazine, Tz).

  2. Effect of TCO loading on nanoparticle binding using BOND-2.
    Figure 2: Effect of TCO loading on nanoparticle binding using BOND-2.

    a, Fluorescence intensity measurements on live cells following sequential incubations with 10 µg ml−1 TCO-modified antibody and 10 nM Tz–MFNP using flow cytometry. Trastuzumab (anti-HER2), cetuximab (anti-EGFR) and anti-EpCAM antibodies were loaded with different numbers of TCO and measured by MALDI-TOF mass spectrometry (Supplementary Figs S1,S2). MFNP targeted HER2 on SK-BR-3 breast cancer cells, EpCAM on HCT 116 colon cancer cells, and EGFR on A549 lung cancer cells. b, Confocal microscopy images of similarly labelled live cells. Control: non-binding, TCO-modified control antibody (clone MOPC-21). HER2 (i,ii); EpCAM (iii,iv); EGFR (v,vi). Scale bar, 50 µm (i).

  3. Comparison of different nanoparticle targeting strategies.
    Figure 3: Comparison of different nanoparticle targeting strategies.

    SK-BR-3, HCT 116 and A549 cells were labelled with different concentrations of MFNP using the two-step BOND-2 or direct MFNP immuno-conjugates, and the fluorescence signal was measured using flow cytometry. MFNP immuno-conjugates were prepared either via maleimide/thiol or TCO/Tz (BOND-1) chemistries. Control samples were incubated with Tz–MFNP only. BOND-2 resulted in significantly higher nanoparticle binding, exceeding the direct immuno-conjugates by a factor of 15 for HER2. b, Fluorescence intensity of SK-BR-3 and HCT 116 cells labelled with 10 µg ml−1 biotin-modified antibody and 100 nM avidin–MFNP was measured using flow cytometry. Biotinylated anti-HER2 and anti-EpCAM antibodies were prepared analogously to the TCO modifications, and biotin levels were determined by MALDI-TOF mass spectrometry (Supplementary Figs S1,S2). Nanoparticle binding increased with biotin loading but remained lower than BOND-2 in both cases. Values for BOND-1 and BOND-2 were taken from a (100 nM MFNP).

  4. Profiling cancer cells using diagnostic magnetic resonance.
    Figure 4: Profiling cancer cells using diagnostic magnetic resonance.

    Magnetic profiling of cell samples (human tumour cell lines: A431, A549, NCI-H1650, HCT 116, SK-BR-3 and SK-OV-3; control: NIH/3T3 fibroblasts, peripheral blood leukocytes) for a panel of cancer markers in scant samples (~1,000 cells) using a recently developed miniaturized diagnostic magnetic resonance device. Cells were labelled with TCO–antibodies followed by Tz–MFNP before measurement of transverse relaxation time (R2). a, Marker expression levels, determined based on the ratio of the positive marker (ΔR2+) and control (ΔR2θ) signals (see Supplementary Methods), were heterogeneous for tumour cells but normal for the control fibroblasts and leukocytes, with the exception of the leukocyte marker CD45. b, The tumour signals showed excellent correlation with measured marker expression levels, as determined independently by flow cytometry (values listed in Supplementary Table S1).

References

  1. Davis, M. E., Chen, Z. G. & Shin, D. M. Nanoparticle therapeutics: an emerging treatment modality for cancer. Nature Rev. Drug Discov. 7, 771782 (2008).
  2. Weissleder, R. & Pittet, M. J. Imaging in the era of molecular oncology. Nature 452, 580589 (2008).
  3. Giljohann, D. A. & Mirkin, C. A. Drivers of biodiagnostic development. Nature 462, 461464 (2009).
  4. Choi, H. S. et al. Design considerations for tumour-targeted nanoparticles. Nature Nanotech. 5, 4247 (2010).
  5. Jin, Y. & Gao, X. Plasmonic fluorescent quantum dots. Nature Nanotech. 4, 571576 (2009).
  6. Chen, Z. et al. Protein microarrays with carbon nanotubes as multicolor Raman labels. Nature Biotechnol. 26, 12851292 (2008).
  7. Qian, X. et al. In vivo tumor targeting and spectroscopic detection with surface-enhanced Raman nanoparticle tags. Nature Biotechnol. 26, 8390 (2008).
  8. Lee, J. H. et al. Artificially engineered magnetic nanoparticles for ultra-sensitive molecular imaging. Nature Med. 13, 9599 (2007).
  9. Gao, X., Cui, Y., Levenson, R. M., Chung, L. W. & Nie, S. In vivo cancer targeting and imaging with semiconductor quantum dots. Nature Biotechnol. 22, 969976 (2004).
  10. Nam, J. M., Thaxton, C. S. & Mirkin, C. A. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science 301, 18841886 (2003).
  11. Peer, D. et al. Nanocarriers as an emerging platform for cancer therapy. Nature Nanotech. 2, 751760 (2007).
  12. Cho, K., Wang, X., Nie, S., Chen, Z. G. & Shin, D. M. Therapeutic nanoparticles for drug delivery in cancer. Clin. Cancer Res. 14, 13101316 (2008).
  13. Akinc, A. et al. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nature Biotechnol. 26, 561569 (2008).
  14. Shaw, S. Y. et al. Perturbational profiling of nanomaterial biologic activity. Proc. Natl Acad. Sci. USA 105, 73877392 (2008).
  15. Xing, Y. et al. Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry. Nature Protoc. 2, 11521165 (2007).
  16. Devaraj, N. K., Upadhyay, R., Haun, J. B., Hilderbrand, S. A. & Weissleder, R. Fast and sensitive pretargeted labeling of cancer cells through a tetrazine/trans-cyclooctene cycloaddition. Angew. Chem. Int. Ed. 48, 70137016 (2009).
  17. Devaraj, N. K., Weissleder, R. & Hilderbrand, S. A. Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjug. Chem. 19, 22972299 (2008).
  18. Blackman, M. L., Royzen, M. & Fox, J. M. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels–Alder reactivity. J. Am. Chem. Soc. 130, 1351813519 (2008).
  19. Lee, H., Sun, E., Ham, D. & Weissleder, R. Chip-NMR biosensor for detection and molecular analysis of cells. Nature Med. 14, 869874 (2008).
  20. Lee, H., Yoon, T. J., Figueiredo, J. L., Swirski, F. K. & Weissleder, R. Rapid detection and profiling of cancer cells in fine-needle aspirates. Proc. Natl Acad. Sci. USA 106, 1245912464 (2009).
  21. Hudis, C. A. Trastuzumab—mechanism of action and use in clinical practice. N. Engl. J. Med. 357, 3951 (2007).
  22. Hong, K. W. et al. A novel anti-EGFR monoclonal antibody inhibiting tumor cell growth by recognizing different epitopes from cetuximab. J. Biotechnol. 145, 8491 (2010).
  23. Troise, F., Cafaro, V., Giancola, C., D'Alessio, G. & De Lorenzo, C. Differential binding of human immunoagents and Herceptin to the ErbB2 receptor. FEBS J. 275, 49674979 (2008).
  24. Green, N. M. Avidin and streptavidin. Methods Enzymol. 184, 5167 (1990).
  25. Chinol, M. et al. Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce immunogenicity. Br. J. Cancer 78, 189197 (1998).
  26. Nagrath, S. et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 450, 12351239 (2007).
  27. Maheswaran, S. et al. Detection of mutations in EGFR in circulating lung-cancer cells. N. Engl. J. Med. 359, 366377 (2008).
  28. Pantel, K., Brakenhoff, R. H. & Brandt, B. Detection, clinical relevance and specific biological properties of disseminating tumour cells. Nature Rev. Cancer 8, 329340 (2008).
  29. Reynolds, F., O'Loughlin, T., Weissleder, R. & Josephson, L. Method of determining nanoparticle core weight. Anal. Chem. 77, 814817 (2005).
  30. Piran, U. & Riordan, W. J. Dissociation rate constant of the biotin–streptavidin complex. J. Immunol. Methods 133, 141143 (1990).

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Affiliations

  1. Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge St, CPZN 5206, Boston, Massachusetts 02114, USA

    • Jered B. Haun,
    • Neal K. Devaraj,
    • Scott A. Hilderbrand,
    • Hakho Lee &
    • Ralph Weissleder
  2. Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA

    • Ralph Weissleder

Contributions

J.B.H. designed and performed the experiments, analysed the data and wrote the manuscript. N.K.D. and S.A.H. developed and synthesized the bioorthogonal chemistries. H.L. performed the magnetic resonance measurements. R.W. provided overall guidance, designed experiments, reviewed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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The authors declare no competing financial interests.

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