Preparation of Heterobivalent and Multivalent Radiopharmaceuticals to Target Tumors Over-Expressing Integrins

  • Guillermina Ferro-Flores
  • Blanca Ocampo-García
  • Clara Santos-Cuevas
  • Nallely Jiménez-Mancilla
  • Myrna Luna-Gutiérrez
  • Flor de M. Ramírez
  • Enrique Morales-Avila
  • Luis M. De León-Rodríguez
  • Erika Azorín-Vega
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Radiolabeled heterobivalent and multivalent molecules that interact concomitantly with two or more target proteins on tumor cells is a strategy for specific, sensitive, and noninvasive tumor imaging and targeted therapy. Many groups have reported the use of radiolabeled peptides based on the Arg-Gly-Asp (RGD) sequence for the in vivo imaging of integrins. However, the in vitro and in vivo efficacy as heterobivalent and multivalent RGD systems for both molecular imaging and targeted radiotherapy (theranostic radiopharmaceuticals) has been scarcely studied. In our research, the design, synthesis, and in vitro characterization of the heterobivalent 99mTc-labeled trans-activator of transcription (49–57)-RGDyK peptide (99mTc-Tat(49–57)-RGDyK) and the multivalent 177Lu-labeled gold nanoparticle-(RGD)100 system (177Lu-AuNP-c[RGDfK(C)]) were first developed. Secondly, the in vivo imaging of tumors over-expressing integrins and the radiation absorbed dose estimations to produce a therapeutic effect by Auger and low-energy electrons from 99mTc internalized in cancer cell nuclei, as well as the effect of beta particles emitted from 177Lu decay, were performed. In this chapter, the following protocols for the developed systems are presented: (1) the synthesis and radiolabeling of heterobivalent RGD peptide and multivalent AuNP-RGDs, (2) the physicochemical and in vitro biochemical characterization of the systems, (3) the radiation absorbed dose assessment, and (4) the in vivo evaluation of the heterobivalent and multivalent radiopharmaceuticals for molecular imaging and targeted radiotherapy. Methods such as immunohistochemical analysis, microSPECT/CT for molecular imaging and pharmacokinetics, microPET/CT for metabolic activity measurement, histological studies, and VEGF gene expression by PCR in tumor tissues were applied for the 177Lu-AuNP-c[RGDfK(C)] therapeutic efficacy assessment.


Tat-RGD Heterobivalent radiopeptides Radiolabeled gold nanoparticles Gold nanoparticles-RGD Theranostic radiopharmaceuticals Auger electrons Lutetium-177 



This study was supported by the Mexican National Council of Science and Technology (CONACYT-SEP-CB-2014-01-242443).


  1. 1.
    Thakur M, Lentle BC (2005) Report of a summit on molecular imaging. Radiology 236:753–755CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ferro-Flores G, Murphy CA, Melendez-Alafort L (2006) Third generation radiopharmaceuticals. Curr Pharm Anal 2:339–352CrossRefGoogle Scholar
  3. 3.
    Buchegger F, Perillo-Adamer F, Dupertuis YM et al (2006) Auger radiation targeted into DNA: a therapy perspective. Eur J Nucl Med Mol Imaging 33:1352–1363CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Zahid M, Robbins PD (2015) Cell-type specific penetrating peptides: therapeutic promises and challenges. Molecules 20:13055–13070CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Torchilin VP (2008) Tat peptide-mediated intracellular delivery of pharmaceutical nanocarriers. Adv Drug Deliv Rev 60:548–558CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Kubas H, Schäfer M, Bauder-Wüst U (2010) Multivalent cyclic RGD ligands: influence of linker lengths on receptor binding. Nucl Med Biol 37:885–891CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Taherian A, Li X, Liu Y et al (2011) Differences in integrin expression and signaling within human breast cancer cells. BMC Cancer 11:1–15CrossRefGoogle Scholar
  8. 8.
    Pointer SM, Muller WJ (2009) Integrins in mammary-stem-cell biology and breast cancer progression – a role in cancer stem cells? Integrins in stem cell and cancer initiation. J Cell Sci 122:207–214CrossRefGoogle Scholar
  9. 9.
    Liu S (2009) Radiolabeled cyclic RGD peptides as integrin α(ν)β(3)-targeted radiotracers: maximizing binding affinity via bivalency. Bioconjug Chem 20:2199–2213CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hao G, Sun X, Do QN et al (2012) Cyclization of RGD peptide sequences via the macrocyclic chelator DOTA for integrin imaging. Dalton Trans 41:14051–14054CrossRefPubMedGoogle Scholar
  11. 11.
    Haubner R, Decristoforo C (2009) Radiolabeled RGD peptides and peptidomimetics for tumor targeting. Front Biosci 14:872–886CrossRefGoogle Scholar
  12. 12.
    Ocampo-Garcia BE, Santos-Cuevas CL, De Leon-Rodriguez L et al (2013) Design and biological evaluation of 99mTc-N2S2-Tat(49-57)-c(RGDyK): a hybrid radiopharmaceutical for tumors expressing α(v)β(3) integrins. Nucl Med Biol 40:481–487CrossRefPubMedGoogle Scholar
  13. 13.
    Ferro-Flores G, Ocampo-Garcia BE, Santos-Cuevas CL et al (2014) Multifunctional radiolabeled nanoparticles for targeted therapy. Curr Med Chem 21:124–138CrossRefPubMedGoogle Scholar
  14. 14.
    Mendoza-Nava H, Ferro-Flores G, Ocampo-Garcia BE et al (2013) Laser heating of gold nanospheres functionalized with octreotide: in vitro effect on HeLa cell viability. Photomed Laser Surg 31:17–22CrossRefPubMedGoogle Scholar
  15. 15.
    Ocampo-Garcia BE, Ferro-Flores G, Morales-Avila E et al (2011) Kit for preparation of multimeric receptor-specific 99mTc-radiopharmaceuticals based on gold nanoparticles. Nucl Med Commun 32:1095–1104CrossRefPubMedGoogle Scholar
  16. 16.
    Jimenez-Mancilla NP, Ferro-Flores G, Ocampo-Garcia BE et al (2012) Multifunctional targeted radiotherapy system for induced tumors expressing gastrin-releasing peptide receptors. Curr Nanosci 18:193–201CrossRefGoogle Scholar
  17. 17.
    Jimenez-Mancilla NP, Ferro-Flores G, Santos-Cuevas CL et al (2013) Multifuctional targeted therapy system based on 99mTc/177Lu-labeled gold nanoparticles-tat(49-57)-Lys3-bombesin internalized in nuclei of prostate cancer cells. J Label Compd Radiopharm 56:663–671CrossRefGoogle Scholar
  18. 18.
    Luna-Gutierrez M, Ferro-Flores G, Ocampo-Garcia BE et al (2013) A therapeutic system of 177Lu-labeled gold nanoparticles-RGD internalized in breast cancer cells. J Mex Chem Soc 57:212–219Google Scholar
  19. 19.
    Luna-Gutierrez M, Ferro-Flores G, Ocampo-Garcia BE et al (2012) 177Lu-labeled monomeric, dimeric and multimeric RGD peptides for the therapy of tumors expressing α(v)β(3) integrins. J Label Compd Radiopharm 55:140–148CrossRefGoogle Scholar
  20. 20.
    Vilchis-Juarez A, Ferro-Flores G, Santos-Cuevas CL et al (2014) Molecular targeting radiotherapy with cyclo-RGDfK(C) peptides conjugated to 177Lu-labeled gold nanoparticles in tumor-bearing mice. J Biomed Nanotechnol 10:393–404CrossRefPubMedGoogle Scholar
  21. 21.
    Miller WH, Hartmann-Siantar C, Fisher D et al (2005) Evaluation of beta-absorbed fractions in a mouse model for 90Y, 188Re, 166Ho, 149Pm, 64Cu, and 177Lu radionuclides. Cancer Biother Radiopharm 20:436–449CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Guillermina Ferro-Flores
    • 1
  • Blanca Ocampo-García
    • 1
  • Clara Santos-Cuevas
    • 1
  • Nallely Jiménez-Mancilla
    • 2
  • Myrna Luna-Gutiérrez
    • 1
  • Flor de M. Ramírez
    • 3
  • Enrique Morales-Avila
    • 4
  • Luis M. De León-Rodríguez
    • 5
  • Erika Azorín-Vega
    • 1
  1. 1.Department of Radioactive MaterialsInstituto Nacional de Investigaciones NuclearesEstado de MéxicoMexico
  2. 2.Instituto Nacional de Investigaciones NuclearesCátedras CONACyTEstado de MéxicoMexico
  3. 3.Department of ChemistryInstituto Nacional de Investigaciones NuclearesEstado de MéxicoMexico
  4. 4.Faculty of ChemistryUniversidad Autónoma del Estado de MéxicoEstado de MéxicoMexico
  5. 5.School of Chemical SciencesThe University of AucklandAucklandNew Zealand

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