Targeted zwitterionic near-infrared fluorophores for improved optical imaging

Journal name:
Nature Biotechnology
Volume:
31,
Pages:
148–153
Year published:
DOI:
doi:10.1038/nbt.2468
Received
Accepted
Published online

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.

At a glance

Figures

  1. Targeted NIR fluorophores and an improved SBR during cell-based assays.
    Figure 1: Targeted NIR fluorophores and an improved SBR during cell-based assays.

    (a) Chemical structure, molecular weight (MW), logD, net surface charge and three-dimensional (3D) modeling of the geometrical positions of the charge and hydrophobicity of cRGD–ZW800-1, cRGD-CW800 and cRGD-Cy5.5 at pH 7.4. Red, negative charge; blue, positive charge; gray, hydrophobicity. (b) Live-cell binding assay for cRGD–ZW800-1, cRGD-CW800 and cRGD-Cy5.5 (top and middle) or NIR fluorophores alone (bottom) in M21 (αvβ3 positive) and M21-L (αvβ3 negative) melanoma cell lines after incubation with 2 μM of each molecule for 30 min at 37 °C. Scale bars, 100 μm. All NIR fluorescence images had identical exposure times and are normalized to the peak signal. (c) Antibody binding assay for secondary antibodies (2′Ab) conjugated with ZW800-1, CW800 and Cy5.5 in MDA-MB-361 (MB-361, HER2 positive) and MDA-MB-231 (MB-231, HER2 negative) human breast cancer cell lines in the presence (top two rows) or absence (bottom two rows) of the HER2 primary antibody (Ab). Scale bars, 50 μm. All NIR fluorescence images had identical exposure times and are normalized to the peak signal.

  2. NIR fluorophore-conjugated antibodies and an improved SBR during histopathological analysis.
    Figure 2: NIR fluorophore–conjugated antibodies and an improved SBR during histopathological analysis.

    (a) H&E (left) and NIR fluorescence immunostaining (right) of prostate tissue using a rabbit human α-methylacyl-CoA racemase (AMACR)-specific primary antibody (Ab; top), goat rabbit-specific secondary (2′Ab) and NIR-conjugated mouse goat-specific tertiary antibodies (3′Ab) alone (middle) and the NIR fluorophores alone (2 μM each; bottom). All NIR fluorescence images had identical exposure times and are normalized to the peak signal. (b) H&E (left) and NIR fluorescence immunostaining (right) of breast tissue using a rabbit human HER2-specific primary antibody (top), goat rabbit-specific secondary and NIR-conjugated mouse goat-specific tertiary antibodies alone (middle) and the NIR fluorophores alone (2 μM each; bottom). All NIR fluorescence images had identical exposure times and are normalized to the peak signal. Scale bars, 200 μm.

  3. Improved in vivo optical imaging using zwitterionic, NIR-fluorescent, targeted small molecules and proteins.
    Figure 3: Improved in vivo optical imaging using zwitterionic, NIR-fluorescent, targeted small molecules and proteins.

    (a) Real-time intraoperative melanoma detection using targeted small molecules. cRGD–ZW800-1 (3 nmol; 100 pmol/g; left), cRGD-CW800 (3 nmol; 100 pmol/g; middle) or cRGD-Cy5.5 (10 nmol; 500 pmol/g; right) was injected intravenously into mice with melanoma tumors. Shown are representative (n = 5 mice per group) color and NIR fluorescence images immediately before injection (0 h pre-injection) and at 4 h after injection (4 h post-injection). T(+) = integrin αvβ3–positive tumor; T(–) = integrin αvβ3–negative tumor; arrows, kidneys; red dotted circle, region of interest (ROI) used for TBR background measurement. Quantifications of the image signal and background are also shown at 4 h after injection (bottom). All NIR fluorescence images had identical exposure times and are normalized to the peak signal. (b) Real-time intraoperative liver (left) and lung (right) tumor detection using targeted small molecules. cRGD–ZW800-1 (10 nmol; top), cRGD-CW800 (10 nmol; middle) or cRGD-Cy5.5 (10 nmol; bottom) was injected intravenously into each mouse 4 h before imaging. Shown are representative (n = 5 mice per group) color images, NIR fluorescence images and a pseudocolored merge of the two. AW, abdominal wall; GB, gallbladder; He, heart; In, intestine; Li, liver; Lu, lungs; St, stomach; TW, thoracic wall. Open arrows indicate tumors. All NIR fluorescence images had identical exposure times and are normalized to the peak signal. (c) Real-time intraoperative thrombus detection using targeted proteins. FBG–ZW800-1 (40 pmol per g body weight; left), FBG-CW800 (40 pmol per g body weight; middle) or FBG-Cy5.5 (40 pmol per g body weight; right) was injected intravenously into Sprague-Dawley rats 1 h after mucosal resection (arrows) in the stomach (shown) or mesenteric vessels (Supplementary Fig. 6). CBRs (bottom graphs) were calculated from the ratio of the signal at the site of injury site to the nearby normal tissue background. Arrows indicate injury sites. Statistical analysis was performed using one-way analysis of variance followed by Tukey's multiple-comparison test (*P < 0.05, **P < 0.01 or ***P < 0.001 for ZW vs. CW, ZW vs. Cy5.5, CW vs. Cy5.5). All NIR fluorescence images had identical exposure times and are normalized to the peak signal. Error bars, mean ± s.d. Scale bars, 1 cm.

  4. Modification of a primary amine by conventional anionic NIR fluorophores (top) and zwitterionic NIR fluorophores (bottom).
    Figure 4: Modification of a primary amine by conventional anionic NIR fluorophores (top) and zwitterionic NIR fluorophores (bottom).

References

  1. Frangioni, J.V. In vivo near-infrared fluorescence imaging. Curr. Opin. Chem. Biol. 7, 626634 (2003).
  2. Gioux, S., Choi, H.S. & Frangioni, J.V. Image-guided surgery using invisible near-infrared light: fundamentals of clinical translation. Mol. Imaging 9, 237255 (2010).
  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, 615 (2010).
  4. Ballou, B. et al. Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies. Cancer Immunol. Immunother. 41, 257263 (1995).
  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, 251257 (1998).
  6. Ye, Y. & Chen, X. Integrin targeting for tumor optical imaging. Theranostics 1, 102126 (2011).
  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, 3948 (2008).
  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, 26202640 (2010).
  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, 892896 (1999).
  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, 34033411 (2003).
  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, 28042807 (2008).
  12. Lee, S., Park, K., Kim, K., Choi, K. & Kwon, I.C. Activatable imaging probes with amplified fluorescent signals. Chem. Commun. (Camb.) 36, 42504260 (2008).
  13. Kobayashi, T. et al. Highly activatable and rapidly releasable caged fluorescein derivatives. J. Am. Chem. Soc. 129, 66966697 (2007).
  14. Urano, Y. et al. Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes. Nat. Med. 15, 104109 (2009).
  15. Choi, H.S. et al. Renal clearance of quantum dots. Nat. Biotechnol. 25, 11651170 (2007).
  16. Choi, H.S. et al. Design considerations for tumour-targeted nanoparticles. Nat. Nanotechnol. 5, 4247 (2010).
  17. Choi, H.S. et al. Rapid translocation of nanoparticles from the lung airspaces to the body. Nat. Biotechnol. 28, 13001303 (2010).
  18. Choi, H.S. & Frangioni, J.V. Nanoparticles for biomedical imaging: fundamentals of clinical translation. Mol. Imaging 9, 291310 (2010).
  19. Choi, H.S. et al. Synthesis and in vivo fate of zwitterionic near-infrared fluorophores. Angew. Chem. Int. Edn Engl. 50, 62586263 (2011).
  20. Hyun, H. et al. cGMP-compatible preparative scale synthesis of near-infrared fluorophores. Contrast Media Mol. Imaging 7, 516524 (2012).
  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, 829841 (1991).
  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, 785792 (2006).
  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, 367382 (1998).
  24. Gitlin, I., Gudiksen, K.L. & Whitesides, G.M. Effects of surface charge on denaturation of bovine carbonic anhydrase. ChemBioChem 7, 12411250 (2006).
  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, 723731 (2009).
  26. Zwaal, R.F., Comfurius, P. & van Deenen, L.L. Membrane asymmetry and blood coagulation. Nature 268, 358360 (1977).
  27. Frangioni, J.V. New technologies for human cancer imaging. J. Clin. Oncol. 26, 40124021 (2008).
  28. Rasmussen, F. Renal clearance: species differences and similarities. Vet. Res. Commun. 7, 301306 (1983).
  29. Reagan-Shaw, S., Nihal, M. & Ahmad, N. Dose translation from animal to human studies revisited. FASEB J. 22, 659661 (2008).
  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, 927930 (1973).
  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, 196211 (2006).
  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, 80098014 (2004).
  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, 29432952 (2009).

Download references

Author information

Affiliations

  1. Department of Medicine, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

    • Hak Soo Choi,
    • Summer L Gibbs,
    • Jeong Heon Lee,
    • Soon Hee Kim,
    • Yoshitomo Ashitate,
    • Fangbing Liu,
    • Hoon Hyun,
    • GwangLi Park,
    • Yang Xie &
    • John V Frangioni
  2. WCU Program, Department of BIN Fusion Technology, Chonbuk National University, Jeonju, South Korea.

    • Soon Hee Kim
  3. Department of Medicine, Cardiovascular Division, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

    • Soochan Bae
  4. Department of Chemistry, Georgia State University, Atlanta, Georgia, USA.

    • Maged Henary
  5. Department of Radiology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

    • John V Frangioni

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.

Competing financial 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.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (2 MB)

    Supplementary Figures 1–6, Supplementary Methods and Supplementary Table 1

Additional data