Molecular Imaging and Biology

, Volume 20, Issue 2, pp 249–259 | Cite as

A Comparison of [99mTc]Duramycin and [99mTc]Annexin V in SPECT/CT Imaging Atherosclerotic Plaques

  • Yan Hu
  • Guobing Liu
  • He Zhang
  • Yanli Li
  • Brian D. Gray
  • Koon Y Pak
  • Hak Soo Choi
  • Dengfeng Cheng
  • Hongcheng Shi
Research Article



Apoptosis is a key factor in unstable plaques. The aim of this study is to evaluate the utility of visualizing atherosclerotic plaques with radiolabeled duramycin and Annexin V.


ApoE−/− mice were fed with a high-fat diet to develop atherosclerosis, C57 mice as a control. Using a routine conjugation protocol, highly pure [99mTc]duramycin and [99mTc]Annexin V were obtained, which were applied for in vitro cell assays of apoptosis and in vivo imaging of atherosclerotic plaques in the animal model. Oil Red O staining, TUNEL, hematoxylin-eosin (HE), and CD68 immunostaining were used to evaluate the deposition of lipids and presence of apoptotic macrophages in the lesions where focal intensity positively correlated with the uptake of both tracers.


[99mTc]duramycin and [99mTc]Annexin V with a high radiochemical purity (97.13 ± 1.52 and 94.94 ± 0.65 %, respectively) and a well stability at room temperature were used. Apoptotic cells binding activity to [99mTc]duramycin (Kd, 6.92 nM and Bmax, 56.04 mol/1019 cells) was significantly greater than [99mTc]Annexin V (Kd, 12.63 nM and Bmax, 31.55 mol/1019 cells). Compared with [99mTc]Annexin V, [99mTc]duramycin bound avidly to atherosclerotic lesions with a higher plaque-to-background ratio (P/B was 8.23 ± 0.91 and 5.45 ± 0.48 at 20 weeks, 15.02 ± 0.23 and 12.14 ± 0.22 at 30 weeks). No plaques were found in C57 control mice. Furthermore, Oil Red O staining showed lipid deposition areas were significantly increased in ApoE−/− mice at 20 and 30 weeks, and TUNEL and CD68 staining confirmed that the focal uptake of both tracers contained abundant apoptotic macrophages.


This stable, fast clearing, and highly specific [99mTc]duramycin, therefore, can be useful for the quantification of vulnerable atherosclerotic plaques.

Key Words

Atherosclerosis Plaque vulnerability Duramycin Annexin V Phosphatidylethanolamine Phosphatidylserine Micro-SPECT/CT 



Funding support from The National Nature Science Foundation of China (81471706, and 81671735) and The Shanghai Science and Technology Committee International Collaboration Project (16410722700) is gratefully appreciated. The authors are grateful to technical supports from Prof. Yingjian Zhang and Dr. Jianping Zhang from Center for Biomedical Imaging, Fudan University, and Shanghai Engineering Research Center of Molecular Imaging Probes.

Author Contributions

Y.H., G.B.L, and D.F.C participated in the experimental and data analysis. Y.H. wrote the manuscript. Y.H., G.B.L, Y.L.L, and H.Z. involved in animal imaging and dealt with the aortas. B.G, K.P, and H.S.C. helped in polishing the articles. Y.H., D.F.C, and H.C.S designed and controlled the quality of study. All authors have reviewed the manuscript.

Compliance with Ethical Standards

Conflict of Interest

Brian D Gray and Koon Y Pak are employees of Molecular Targeting Technologies, Inc. All other authors declare that there is no conflict of interest regarding the publication of this paper. At all times, the other authors had full control over study data and interpretation.


  1. 1.
    Joshi NV, Vesey AT, Williams MC et al (2014) [18F]fluoride positron emission tomography for identification of ruptured and high-risk coronary atherosclerotic plaques: a prospective clinical trial. Lancet 383:705–713CrossRefPubMedGoogle Scholar
  2. 2.
    van Tilborg GAF, Vucic E, Strijkers GJ et al (2010) Annexin A5-functionalized bimodal nanoparticles for MRI and fluorescence imaging of atherosclerotic plaques. Biocongate Chem 21:1794–1803CrossRefGoogle Scholar
  3. 3.
    Leung K (2004) [99mTc]Tricarbonyl-His6-annexin A5.
  4. 4.
    Tepper CG, Studzinski GP (1993) Resistance of mitochondrial DNA to degradation characterizes the apoptotic but not the necrotic mode of human leukemia cell death. J Cell Biochem 52:352–361CrossRefPubMedGoogle Scholar
  5. 5.
    Knezevic T, Myers VD, Gordon J et al (2015) BAG3: a new player in the heart failure paradigm. Heart Fail Rev 20:423–434CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    De Saint-Hubert M, Bauwens M, Deckers N et al (2014) In vivo molecular imaging of apoptosis and necrosis in atherosclerotic plaques using microSPECT-CT and microPET-CT imaging. Mol Imaging Biol 16:246–254CrossRefPubMedGoogle Scholar
  7. 7.
    Figg NL, Bennett MR (2015) Quantification of apoptosis in mouse atherosclerotic lesions. Methods Mol Biol 1339:191–199CrossRefPubMedGoogle Scholar
  8. 8.
    Daleke DL (2003) Regulation of transbilayer plasma membrane phospholipid asymmetry. J Lipid Res 44:233–242CrossRefPubMedGoogle Scholar
  9. 9.
    Johnson SE, Li Z, Liu Y et al (2013) Whole-body imaging of high-dose ionizing irradiation-induced tissue injuries using [99mTc]duramycin. J Nucl Med 54:1397–1403CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zhao M (2011) Lantibiotics as probes for phosphatidylethanolamine. Amino Acids 41:1071–1079CrossRefPubMedGoogle Scholar
  11. 11.
    Isobe S, Tsimikas S, Zhou J et al (2006) Noninvasive imaging of atherosclerotic lesions in apolipoprotein E-deficient and low-density-lipoprotein receptor-deficient mice with annexin A5. J Nucl Med 47:1497–1505PubMedGoogle Scholar
  12. 12.
    Subbarayan M, Hafeli UO, Feyes DK et al (2003) A simplified method for preparation of [99mTc]annexin V and its biologic evaluation for in vivo imaging of apoptosis after photodynamic therapy. J Nucl Med 44(4):650–656PubMedGoogle Scholar
  13. 13.
    Benali K, Louedec L, Azzouna R B, et al. (2014) Preclinical validation of [99mTc]annexin A5-128 in experimental autoimmune myocarditis and infective endocarditis: comparison with [99mTc]HYNIC-annexin A5. Mol Imaging 13Google Scholar
  14. 14.
    Lu C, Jiang Q, Hu M et al (2013) Preliminary biological evaluation of novel [99mTc]Cys-Annexin A5 as a apoptosis imaging agent. Molecules 18:6908–6918CrossRefPubMedGoogle Scholar
  15. 15.
    Cheng D, Li X, Zhang C et al (2015) Detection of vulnerable atherosclerosis plaques with a dual-modal single-photon-emission computed tomography/magnetic resonance imaging probe targeting apoptotic macrophages. ACS Appl Mater Interfaces 7:2847–2855CrossRefPubMedGoogle Scholar
  16. 16.
    Burgmaier M, Schutters K, Willems B et al (2014) AnxA5 reduces plaque inflammation of advanced atherosclerotic lesions in apoE−/− mice. J Cell Mol Med 18:2117–2124CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Zhao Y, Watanabe A, Zhao S et al (2014) Suppressive effects of irbesartan on inflammation and apoptosis in atherosclerotic plaques of apoE−/− mice: molecular imaging with [14C]FDG and [99mTc]Annexin A5. PLoS One 9:e89338CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Ogawa K, Ohtsuki K, Shibata T et al (2013) Development and evaluation of a novel Tc-99m labeled Annexin A5 for early detection of response to chemotherapy. PLoS One 8:e81191CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Tian R, Pan D (2012) Imaging myocardial ischemia and reperfusion injury via Cy5.5-Annexin V. Nucl Med Mol Imaging 46:155–161CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Li X, Wang C, Tan H et al (2016) Gold nanoparticles-based SPECT/CT imaging probe targeting for vulnerable atherosclerosis plaques. Biomaterials 108:71–80CrossRefPubMedGoogle Scholar
  21. 21.
    Zhao M, Li Z, Bugenhagen S (2008) Tc-99m labeled duramycin as a novel phosphatidylethanolamine-binding molecular probe. J Nucl Med 49:1345–1352CrossRefPubMedGoogle Scholar
  22. 22.
    Elvas F, Vangestel C, Rapic S et al (2015) Characterization of [99mTc]duramycin as a SPECT imaging agent for early assessment of tumor apoptosis. Mol Imaging Biol 17:838–847CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Zhang Y, Stevenson GD, Barber C et al (2013) Imaging of rat cerebral ischemia-reperfusion injury using Tc-99m labeled duramycin. Nucl Med Biol 40:80–88CrossRefPubMedGoogle Scholar
  24. 24.
    Liu Z, Larsen BT, Lerman LO et al (2016) Detection of atherosclerotic plaques in ApoE-deficient mice using [99mTc]duramycin. Nucl Med Biol 43:496–505CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Wang Y, Liu G, Hnatowich DJ (2006) Methods for MAG3 conjugation and Tc-99m radiolabeling of biomolecules. Nat Protoc 1:1477–1480CrossRefPubMedGoogle Scholar
  26. 26.
    Wang Y, Liu X, Hnatowich DJ (2007) An improved synthesis of NHS-MAG3 for conjugation and radiolabeling of biomolecules with Tc-99m at room temperature. Nat Protoc 2:972–978CrossRefPubMedGoogle Scholar
  27. 27.
    Kim MJ, Jeong HJ, Kim DW et al (2014) PEP-1-PON1 protein regulates inflammatory response in raw 264.7 macrophages and ameliorates inflammation in a TPA-induced animal model. PLoS ONE 9:e86034CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Laochumroonvorapong P, Paul S, Elkon KB et al (1996) H2O2 induces monocyte apoptosis and reduces viability of Mycobacterium avium-M. intracellulare within cultured human monocytes. Infect Immun 64:452–459PubMedPubMedCentralGoogle Scholar
  29. 29.
    Tyurin VA, Balasubramanian K, Winnica D et al (2014) Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic ‘eat-me’ signals: cleavage and inhibition of phagocytosis by Lp-PLA2. Cell Death Differ 21:825–835CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Audi S, Li Z, Capacete J et al (2012) Understanding the in vivo uptake kinetics of a phosphatidylethanolamine-binding agent [99mTc]duramycin. Nucl Med Biol 39:821–825CrossRefPubMedGoogle Scholar
  31. 31.
    Fadeel B, Xue D (2009) The ins and outs of phospholipid asymmetry in the plasma membrane: roles in health and disease. Crit Rev Biochem Mol 44:264–277CrossRefGoogle Scholar
  32. 32.
    Post JA, Verkleij AJ, Langer GA (1995) Organization and function of sarcolemmal phospholipids in control and ischemic/reperfused cardiomyocytes. J Mol Cell Cardiol 27:749–760CrossRefPubMedGoogle Scholar
  33. 33.
    Callahan MK, Williamson P, Schlegel RA (2000) Surface expression of phosphatidylserine on macrophages is required for phagocytosis of apoptotic thymocytes. Cell Death Differ 7:645–653CrossRefPubMedGoogle Scholar
  34. 34.
    Ogawa K, Aoki M (2014) Radiolabeled apoptosis imaging agents for early detection of response to therapy. Sci World J 2014:1–11CrossRefGoogle Scholar
  35. 35.
    Marconescu A, Thorpe PE (2008) Coincident exposure of phosphatidylethanolamine and anionic phospholipids on the surface of irradiated cells. BBA-Biomembranes 1778:2217–2224CrossRefPubMedGoogle Scholar
  36. 36.
    Zhao M, Li Z (2012) A single-step kit formulation for the Tc-99m labeling of HYNIC-duramycin. Nucl Med Biol 39:1006–1011CrossRefPubMedGoogle Scholar
  37. 37.
    Zhang R, Lu W, Wen X et al (2011) Annexin A5-conjugated polymeric micelles for dual SPECT and optical detection of apoptosis. J Nucl Med 52:958–964CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Wang L, Wang F, Fang W et al (2015) The feasibility of imaging myocardial ischemic/reperfusion injury using Tc-99m labeled duramycin in a porcine model. Nucl Med Biol 42:198–204CrossRefPubMedGoogle Scholar
  39. 39.
    Ke S, Wen X, Wu Q et al (2004) Imaging taxane-induced tumor apoptosis using PEGylated, in-111 labeled Annexin V. J Nucl Med 45:108PubMedGoogle Scholar
  40. 40.
    Glaser M, Collingridge DR, Aboagye EO et al (2003) Iodine-124 labeled annexin-V as a potential radiotracer to study apoptosis using positron emission tomography. Appl Radiat Isot 58:55–62CrossRefPubMedGoogle Scholar

Copyright information

© World Molecular Imaging Society 2017

Authors and Affiliations

  • Yan Hu
    • 1
    • 2
    • 3
  • Guobing Liu
    • 1
    • 2
    • 3
  • He Zhang
    • 1
    • 2
    • 3
  • Yanli Li
    • 1
    • 2
    • 3
  • Brian D. Gray
    • 4
  • Koon Y Pak
    • 4
  • Hak Soo Choi
    • 5
  • Dengfeng Cheng
    • 1
    • 2
    • 3
  • Hongcheng Shi
    • 1
    • 2
    • 3
  1. 1.Department of Nuclear Medicine, Zhongshan HospitalFudan UniversityShanghaiPeople’s Republic of China
  2. 2.Shanghai Institute of Medical ImagingShanghaiChina
  3. 3.Institute of Nuclear MedicineFudan UniversityShanghaiChina
  4. 4.Molecular Targeting Technologies, Inc.West ChesterUSA
  5. 5.Gordon Center for Medical Imaging, Department of RadiologyMassachusetts General Hospital and Harvard Medical SchoolBostonUSA

Personalised recommendations