Development of radiolabeled bis(zinc(II)-dipicolylamine) complexes for cell death imaging

  • Miho Aoki
  • Akira Odani
  • Kazuma OgawaEmail author
Original Article



Although it has been traditionally surmised that phosphatidylserine (PS) externalization is a hallmark of apoptosis, most other non-apoptotic modes of cell death, such as necrosis, are also associated with PS externalization. Bis(zinc-dipicolylamine) (ZnDPA) complexes have been reported to exhibit affinity for PS. The present study aimed to develop novel radiolabeled ZnDPA derivatives for cell death imaging in tumor after treatment with anticancer drugs.


[125I]IB-EG2-ZnDPA and [99mTc]Tc-MAG3-EG2-ZnDPA were designed and prepared. The stabilities of these radiotracers were determined in 0.1 M phosphate buffer (pH 7.4) or murine plasma at 37 °C, and their 1-octanol/water partition coefficients (logP) were measured. The uptake of radioactivity in cancer cells, which were preincubated in a normal medium or in a medium containing 5-FU, was measured after incubation with radiotracers. Accumulation of [99mTc]Tc-MAG3-EG2-ZnDPA in the tumor was evaluated in tumor-bearing mice treated with or without 5-FU, and then TUNEL staining was performed to detect dead cells in the tumor tissue sections.


The radiochemical purities of [125I]IB-EG2-ZnDPA and [99mTc]Tc-MAG3-EG2-ZnDPA exceeded 95%. Although [125I]IB-EG2-ZnDPA gradually decomposing with time, more than 90% of [99mTc]Tc-MAG3-EG2-ZnDPA remained in its intact form in phosphate buffer through 6 h of incubation. Neither [125I]IB-EG2-ZnDPA nor [99mTc]Tc-MAG3-EG2-ZnDPA decomposed so much after 6-h incubation in murine plasma. [125I]IB-EG2-ZnDPA could not specifically recognize PS on the cell surface because of its high lipophilicity. Conversely, [99mTc]Tc-MAG3-EG2-ZnDPA accumulated in cancer cells after treatment with an anticancer drug both in vitro and in vivo, and its accumulation was correlated with the number of TUNEL-positive cells. However, the biodistribution of [99mTc]Tc-MAG3-EG2-ZnDPA was not suitable for imaging because of its low accumulation in tumor and high uptake in abdomen organs.


[99mTc]Tc-MAG3-EG2-ZnDPA could be useful for the early detection of treatment effects after chemotherapy. Since the signal-to-noise ratio is not enough for single photon emission computed tomography imaging, further modification is needed to improve its biodistribution and affinity for PS.


Zinc(II)-dipicolylamine Phosphatidylserine Cell death imaging Technetium 



The authors would like to thank Enago for the English language review.

Supplementary material

12149_2019_1339_MOESM1_ESM.docx (95 kb)
Supplementary material 1 (DOCX 95 KB)


  1. 1.
    Brown JM, Attardi LD. The role of apoptosis in cancer development and treatment response. Nat Rev Cancer. 2005;5(3):231–7.CrossRefGoogle Scholar
  2. 2.
    Ogawa K, Aoki M. Radiolabeled apoptosis imaging agents for early detection of response to therapy. Sci World J. 2014;2014:732603.CrossRefGoogle Scholar
  3. 3.
    Reshef A, Shirvan A, Akselrod-Ballin A, Wall A, Ziv I. Small-molecule biomarkers for clinical PET imaging of apoptosis. J Nucl Med. 2010;51(6):837–40.CrossRefGoogle Scholar
  4. 4.
    Blankenberg FG. In vivo detection of apoptosis. J Nucl Med. 2008;49(Suppl 2):81S–95S.CrossRefGoogle Scholar
  5. 5.
    Smith BA, Smith BD. Biomarkers and molecular probes for cell death imaging and targeted therapeutics. Bioconjug Chem. 2012;23(10):1989–2006.CrossRefGoogle Scholar
  6. 6.
    Hanshaw RG, Smith BD. New reagents for phosphatidylserine recognition and detection of apoptosis. Bioorg Med Chem. 2005;13(17):5035–42.CrossRefGoogle Scholar
  7. 7.
    Blankenberg FG, Strauss HW. Recent advances in the molecular imaging of programmed cell death: part I—pathophysiology and radiotracers. J Nucl Med. 2012;53(11):1659–62.CrossRefGoogle Scholar
  8. 8.
    Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med. 1995;182(5):1545–56.CrossRefGoogle Scholar
  9. 9.
    Tait JF. Imaging of apoptosis. J Nucl Med. 2008;49(10):1573–6.CrossRefGoogle Scholar
  10. 10.
    Schlegel RA, Williamson P. Phosphatidylserine, a death knell. Cell Death Differ. 2001;8:551.CrossRefGoogle Scholar
  11. 11.
    Corsten MF, Hofstra L, Narula J, Reutelingsperger CP. Counting heads in the war against cancer: defining the role of annexin A5 imaging in cancer treatment and surveillance. Cancer Res. 2006;66(3):1255–60.CrossRefGoogle Scholar
  12. 12.
    Lahorte CM, Vanderheyden JL, Steinmetz N, Van de Wiele C, Dierckx RA, Slegers G. Apoptosis-detecting radioligands: current state of the art and future perspectives. Eur J Nucl Med Mol Imaging. 2004;31(6):887–919.CrossRefGoogle Scholar
  13. 13.
    Laufer EM, Reutelingsperger CP, Narula J, Hofstra L. Annexin A5: an imaging biomarker of cardiovascular risk. Basic Res Cardiol. 2008;103(2):95–104.CrossRefGoogle Scholar
  14. 14.
    Jiang X, Li H. MiR-1180-5p regulates apoptosis of Wilms’ tumor by targeting p73. OncoTargets Ther. 2018;11:823–31.CrossRefGoogle Scholar
  15. 15.
    Ohtsuki K, Akashi K, Aoka Y, Blankenberg FG, Kopiwoda S, Tait JF, et al. Technetium-99m HYNIC-annexin V: a potential radiopharmaceutical for the in-vivo detection of apoptosis. Eur J Nucl Med. 1999;26(10):1251–8.CrossRefGoogle Scholar
  16. 16.
    Doue T, Ohtsuki K, Ogawa K, Ueda M, Azuma A, Saji H, et al. Cardioprotective effects of erythropoietin in rats subjected to ischemia-reperfusion injury: assessment of infarct size with 99mTc-annexin V. J Nucl Med. 2008;49(10):1694–700.CrossRefGoogle Scholar
  17. 17.
    Rottey S, Slegers G, Van Belle S, Goethals I, Van de Wiele C. Sequential 99mTc-hydrazinonicotinamide-annexin V imaging for predicting response to chemotherapy. J Nucl Med. 2006;47(11):1813–8.Google Scholar
  18. 18.
    Ogawa K, Ohtsuki K, Shibata T, Aoki M, Nakayama M, Kitamura Y, et al. Development and evaluation of a novel 99mTc-labeled annexin A5 for early detection of response to chemotherapy. PLoS One. 2013;8(12):e81191.CrossRefGoogle Scholar
  19. 19.
    Wang H, Tang X, Tang G, Huang T, Liang X, Hu K, et al. Noninvasive positron emission tomography imaging of cell death using a novel small-molecule probe, (18)F labeled bis(zinc(II)-dipicolylamine) complex. Apoptosis. 2013;18(8):1017–27.CrossRefGoogle Scholar
  20. 20.
    Koulov AV, Stucker KA, Lakshmi C, Robinson JP, Smith BD. Detection of apoptotic cells using a synthetic fluorescent sensor for membrane surfaces that contain phosphatidylserine. Cell Death Differ. 2003;10(12):1357–9.CrossRefGoogle Scholar
  21. 21.
    Hanshaw RG, Lakshmi C, Lambert TN, Johnson JR, Smith BD. Fluorescent detection of apoptotic cells by using zinc coordination complexes with a selective affinity for membrane surfaces enriched with phosphatidylserine. ChemBioChem. 2005;6(12):2214–20.CrossRefGoogle Scholar
  22. 22.
    Kwong JM, Hoang C, Dukes RT, Yee RW, Gray BD, Pak KY, et al. Bis(zinc-dipicolylamine), Zn-DPA, a new marker for apoptosis. Investig Ophthalmol Vis Sci. 2014;55(8):4913–21.CrossRefGoogle Scholar
  23. 23.
    Plaunt AJ, Harmatys KM, Wolter WR, Suckow MA, Smith BD. Library synthesis, screening, and discovery of modified zinc(II)-bis(dipicolylamine) probe for enhanced molecular imaging of cell death. Bioconjug Chem. 2014;25(4):724–37.CrossRefGoogle Scholar
  24. 24.
    Niu G, Chen X. Apoptosis imaging: beyond annexin V. J Nucl Med. 2010;51(11):1659–62.CrossRefGoogle Scholar
  25. 25.
    Lakshmi C, Hanshaw RG, Smith BD. Fluorophore-linked zinc(II)dipicolylamine coordination complexes as sensors for phosphatidylserine-containing membranes. Tetrahedron. 2004;60(49):11307–15.CrossRefGoogle Scholar
  26. 26.
    Ogawa K, Mukai T, Arano Y, Ono M, Hanaoka H, Ishino S, et al. Development of a rhenium-186-labeled MAG3-conjugated bisphosphonate for the palliation of metastatic bone pain based on the concept of bifunctional radiopharmaceuticals. Bioconjug Chem. 2005;16(4):751–7.CrossRefGoogle Scholar
  27. 27.
    Ogawa K, Takeda T, Yokokawa M, Yu J, Makino A, Kiyono Y, et al. Comparison of radioiodine- or radiobromine-labeled RGD peptides between direct and indirect labeling methods. Chem Pharm Bull (Tokyo). 2018;66(6):651–9.CrossRefGoogle Scholar
  28. 28.
    Ogawa K, Mukai T, Inoue Y, Ono M, Saji H. Development of a novel 99mTc-chelate-conjugated bisphosphonate with high affinity for bone as a bone scintigraphic agent. J Nucl Med. 2006;47(12):2042–7.Google Scholar
  29. 29.
    Ogawa K, Shiba K, Akhter N, Yoshimoto M, Washiyama K, Kinuya S, et al. Evaluation of radioiodinated vesamicol analogs for sigma receptor imaging in tumor and radionuclide receptor therapy. Cancer Sci. 2009;100(11):2188–92.CrossRefGoogle Scholar
  30. 30.
    Deutsch E, Libson K, Vanderheyden JL, Ketring AR, Maxon HR. The chemistry of rhenium and technetium as related to the use of isotopes of these elements in therapeutic and diagnostic nuclear medicine. Int J Radiat Appl Instrum Part B Nucl Med Biol. 1986;13(4):465–77.CrossRefGoogle Scholar
  31. 31.
    Ojida A, Mito-oka Y, Sada K, Hamachi I. Molecular recognition and fluorescence sensing of monophosphorylated peptides in aqueous solution by bis(zinc(II)-dipicolylamine)-based artificial receptors. J Am Chem Soc. 2004;126(8):2454–63.CrossRefGoogle Scholar
  32. 32.
    Hu Q, Gao M, Feng G, Chen X, Liu B. A cell apoptosis probe based on fluorogen with aggregation induced emission characteristics. ACS Appl Mater Interfaces. 2015;7(8):4875–82.CrossRefGoogle Scholar
  33. 33.
    Wyffels L, Gray BD, Barber C, Moore SK, Woolfenden JM, Pak KY, et al. Synthesis and preliminary evaluation of radiolabeled bis(zinc(II)-dipicolylamine) coordination complexes as cell death imaging agents. Bioorg Med Chem. 2011;19(11):3425–33.CrossRefGoogle Scholar
  34. 34.
    Rice DR, Plaunt AJ, Turkyilmaz S, Smith M, Wang Y, Rusckowski M, et al. Evaluation of [111In]-labeled zinc-dipicolylamine tracers for SPECT imaging of bacterial infection. Mol Imaging Biol. 2015;17(2):204–13.CrossRefGoogle Scholar

Copyright information

© The Japanese Society of Nuclear Medicine 2019

Authors and Affiliations

  1. 1.Graduate School of Medical SciencesKanazawa UniversityKanazawaJapan
  2. 2.Institute for Frontier Science InitiativeKanazawa UniversityKanazawaJapan

Personalised recommendations