Targeted zwitterionic near-infrared fluorophores for improved optical imaging

Abstract

The signal-to-background ratio (SBR) is the key determinant of sensitivity, detectability and linearity in optical imaging. As signal strength is often constrained by fundamental limits, background reduction becomes an important approach for improving the SBR. We recently reported that a zwitterionic near-infrared (NIR) fluorophore, ZW800-1, exhibits low background. Here we show that this fluorophore provides a much-improved SBR when targeted to cancer cells or proteins by conjugation with a cyclic RGD peptide, fibrinogen or antibodies. ZW800-1 outperforms the commercially available NIR fluorophores IRDye800-CW and Cy5.5 in vitro for immunocytometry, histopathology and immunoblotting and in vivo for image-guided surgery. In tumor model systems, a tumor-to-background ratio of 17.2 is achieved at 4 h after injection of ZW800-1 conjugated to cRGD compared to ratios of 5.1 with IRDye800-CW and 2.7 with Cy5.5. Our results suggest that introducing zwitterionic properties into targeted fluorophores may be a general strategy for improving the SBR in diagnostic and therapeutic applications.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Targeted NIR fluorophores and an improved SBR during cell-based assays.
Figure 2: NIR fluorophore–conjugated antibodies and an improved SBR during histopathological analysis.
Figure 3: Improved in vivo optical imaging using zwitterionic, NIR-fluorescent, targeted small molecules and proteins.
Figure 4

References

  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

    Gioux, S., Choi, H.S. & Frangioni, J.V. Image-guided surgery using invisible near-infrared light: fundamentals of clinical translation. Mol. Imaging 9, 237–255 (2010).

    CAS  Article  Google Scholar 

  3. 3

    Te Velde, E.A., Veerman, T., Subramaniam, V. & Ruers, T. The use of fluorescent dyes and probes in surgical oncology. Eur. J. Surg. Oncol. 36, 6–15 (2010).

    CAS  Article  Google Scholar 

  4. 4

    Ballou, B. et al. Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies. Cancer Immunol. Immunother. 41, 257–263 (1995).

    CAS  Article  Google Scholar 

  5. 5

    Ballou, B. et al. Cyanine fluorochrome-labeled antibodies in vivo: assessment of tumor imaging using Cy3, Cy5, Cy5.5, and Cy7. Cancer Detect. Prev. 22, 251–257 (1998).

    CAS  Article  Google Scholar 

  6. 6

    Ye, Y. & Chen, X. Integrin targeting for tumor optical imaging. Theranostics 1, 102–126 (2011).

    CAS  Article  Google Scholar 

  7. 7

    Tanaka, E. et al. Real-time intraoperative assessment of the extrahepatic bile ducts in rats and pigs using invisible near-infrared fluorescent light. Surgery 144, 39–48 (2008).

    Article  Google Scholar 

  8. 8

    Kobayashi, H., Ogawa, M., Alford, R., Choyke, P.L. & Urano, Y. New strategies for fluorescent probe design in medical diagnostic imaging. Chem. Rev. 110, 2620–2640 (2010).

    CAS  Article  Google Scholar 

  9. 9

    Tung, C.H., Bredow, S., Mahmood, U. & Weissleder, R. Preparation of a cathepsin D sensitive near-infrared fluorescence probe for imaging. Bioconjug. Chem. 10, 892–896 (1999).

    CAS  Article  Google Scholar 

  10. 10

    Achilefu, S. et al. Novel bioactive and stable neurotensin peptide analogues capable of delivering radiopharmaceuticals and molecular beacons to tumors. J. Med. Chem. 46, 3403–3411 (2003).

    CAS  Article  Google Scholar 

  11. 11

    Lee, S. et al. A near-infrared-fluorescence-quenched gold-nanoparticle imaging probe for in vivo drug screening and protease activity determination. Angew. Chem. Int. Edn Engl. 47, 2804–2807 (2008).

    CAS  Article  Google Scholar 

  12. 12

    Lee, S., Park, K., Kim, K., Choi, K. & Kwon, I.C. Activatable imaging probes with amplified fluorescent signals. Chem. Commun. (Camb.) 36, 4250–4260 (2008).

    Article  Google Scholar 

  13. 13

    Kobayashi, T. et al. Highly activatable and rapidly releasable caged fluorescein derivatives. J. Am. Chem. Soc. 129, 6696–6697 (2007).

    CAS  Article  Google Scholar 

  14. 14

    Urano, Y. et al. Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes. Nat. Med. 15, 104–109 (2009).

    CAS  Article  Google Scholar 

  15. 15

    Choi, H.S. et al. Renal clearance of quantum dots. Nat. Biotechnol. 25, 1165–1170 (2007).

    CAS  Article  Google Scholar 

  16. 16

    Choi, H.S. et al. Design considerations for tumour-targeted nanoparticles. Nat. Nanotechnol. 5, 42–47 (2010).

    CAS  Article  Google Scholar 

  17. 17

    Choi, H.S. et al. Rapid translocation of nanoparticles from the lung airspaces to the body. Nat. Biotechnol. 28, 1300–1303 (2010).

    CAS  Article  Google Scholar 

  18. 18

    Choi, H.S. & Frangioni, J.V. Nanoparticles for biomedical imaging: fundamentals of clinical translation. Mol. Imaging 9, 291–310 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Choi, H.S. et al. Synthesis and in vivo fate of zwitterionic near-infrared fluorophores. Angew. Chem. Int. Edn Engl. 50, 6258–6263 (2011).

    CAS  Article  Google Scholar 

  20. 20

    Hyun, H. et al. cGMP-compatible preparative scale synthesis of near-infrared fluorophores. Contrast Media Mol. Imaging 7, 516–524 (2012).

    CAS  Article  Google Scholar 

  21. 21

    Adams, J.C. & Watt, F.M. Expression of β1, β3, β4, and β5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes. J. Cell Biol. 115, 829–841 (1991).

    CAS  Article  Google Scholar 

  22. 22

    Ohnishi, S., Garfein, E.S., Karp, S.J. & Frangioni, J.V. Radiolabeled and near-infrared fluorescent fibrinogen derivatives create a system for the identification and repair of obscure gastrointestinal bleeding. Surgery 140, 785–792 (2006).

    Article  Google Scholar 

  23. 23

    Colton, I.J., Carbeck, J.D., Rao, J. & Whitesides, G.M. Affinity capillary electrophoresis: a physical-organic tool for studying interactions in biomolecular recognition. Electrophoresis 19, 367–382 (1998).

    CAS  Article  Google Scholar 

  24. 24

    Gitlin, I., Gudiksen, K.L. & Whitesides, G.M. Effects of surface charge on denaturation of bovine carbonic anhydrase. ChemBioChem 7, 1241–1250 (2006).

    CAS  Article  Google Scholar 

  25. 25

    Bourré, L., Giuntini, F., Eggleston, I.M., Wilson, M. & MacRobert, A.J. Protoporphyrin IX enhancement by 5-aminolaevulinic acid peptide derivatives and the effect of RNA silencing on intracellular metabolism. Br. J. Cancer 100, 723–731 (2009).

    Article  Google Scholar 

  26. 26

    Zwaal, R.F., Comfurius, P. & van Deenen, L.L. Membrane asymmetry and blood coagulation. Nature 268, 358–360 (1977).

    CAS  Article  Google Scholar 

  27. 27

    Frangioni, J.V. New technologies for human cancer imaging. J. Clin. Oncol. 26, 4012–4021 (2008).

    Article  Google Scholar 

  28. 28

    Rasmussen, F. Renal clearance: species differences and similarities. Vet. Res. Commun. 7, 301–306 (1983).

    CAS  Article  Google Scholar 

  29. 29

    Reagan-Shaw, S., Nihal, M. & Ahmad, N. Dose translation from animal to human studies revisited. FASEB J. 22, 659–661 (2008).

    CAS  Article  Google Scholar 

  30. 30

    Kelley, K.W., Curtis, S.E., Marzan, G.T., Karara, H.M. & Anderson, C.R. Body surface area of female swine. J. Anim. Sci. 36, 927–930 (1973).

    CAS  Article  Google Scholar 

  31. 31

    Humblet, V., Misra, P. & Frangioni, J.V. An HPLC/mass spectrometry platform for the development of multimodality contrast agents and targeted therapeutics: prostate-specific membrane antigen small molecule derivatives. Contrast Media Mol. Imaging 1, 196–211 (2006).

    CAS  Article  Google Scholar 

  32. 32

    Chen, X., Conti, P.S. & Moats, R.A. In vivo near-infrared fluorescence imaging of integrin αvβ3 in brain tumor xenografts. Cancer Res. 64, 8009–8014 (2004).

    CAS  Article  Google Scholar 

  33. 33

    Troyan, S.L. et al. The FLARE™ intraoperative near-infrared fluorescence imaging system: a first-in-human clinical trial in breast cancer sentinel lymph node mapping. Ann. Surg. Oncol. 16, 2943–2952 (2009).

    Article  Google Scholar 

Download references

Acknowledgements

We thank S. Gioux, R. Oketokoun and A. Stockdale for assistance with development of the FLARE (Fluorescence-Assisted Resection and Exploration) imaging system and software. We also thank D. Burrington Jr. for manuscript editing and E. Trabucchi for administrative assistance. This study was supported by the following grants from the US National Institutes of Health: National Cancer Institute Bioengineering Research Partnership grant #R01-CA-115296 (J.V.F.) and National Institute of Biomedical Imaging and Bioengineering grants #R01-EB-010022 and #R01-EB-011523 (both to H.S.C. and J.V.F.); this study was also supported by the Dana Foundation Program in Brain and Immuno-Imaging (H.S.C.). S.H.K. was supported by a WCU Program (R31-20029) from the Korea Ministry of Education, Science and Technology (KMEST).

Author information

Affiliations

Authors

Contributions

H.S.C., S.L.G., S.H.K., Y.A., J.H.L., H.H., G.P., Y.X., F.L., S.B. and M.H. performed the experiments. H.S.C., S.L.G. and J.V.F. reviewed, analyzed and interpreted the data. H.S.C., S.L.G. and J.V.F. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to John V Frangioni.

Ethics declarations

Competing interests

FLARE technology is owned by Beth Israel Deaconess Medical Center, a teaching hospital of Harvard Medical School. It has been licensed to the FLARE Foundation, a nonprofit organization focused on promoting the dissemination of medical imaging technology for research and clinical use. J.V.F. is the founder and chairman of the FLARE Foundation. The Beth Israel Deaconess Medical Center will receive royalties for the sale of FLARE Technology. J.V.F. has elected to surrender post-market royalties to which he would otherwise be entitled as inventor, and has elected to donate pre-market proceeds to the FLARE Foundation. J.V.F. has started three for-profit companies, Curadel, Curadel Medical Devices and Curadel In Vivo Diagnostics, which may someday be nonexclusive sublicensees of FLARE technology.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Methods and Supplementary Table 1 (PDF 2382 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Choi, H., Gibbs, S., Lee, J. et al. Targeted zwitterionic near-infrared fluorophores for improved optical imaging. Nat Biotechnol 31, 148–153 (2013). https://doi.org/10.1038/nbt.2468

Download citation

Further reading

Search

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