Molecular Imaging and Biology

, Volume 13, Issue 2, pp 332–341 | Cite as

In Vitro and Initial In Vivo Evaluation of 68Ga-Labeled Transferrin Receptor (TfR) Binding Peptides as Potential Carriers for Enhanced Drug Transport into TfR Expressing Cells

  • Carmen WänglerEmail author
  • Dina Nada
  • Georg Höfner
  • Simone Maschauer
  • Björn Wängler
  • Stephan Schneider
  • Esther Schirrmacher
  • Klaus T. Wanner
  • Ralf SchirrmacherEmail author
  • Olaf Prante
Research Article



The transferrin receptor (TfR) is one of the most attractive targets to overcome the blood–brain barrier (BBB). It has recently been shown that THRPPMWSPVWP binds to the TfR and is subsequently internalized into TfR-expressing cells. Here, we evaluated the ability of THRPPMWSPVWP to become internalized into human TfR-expressing cells via endocytosis to determine its potential to act as a carrier system for the transport of small molecules across the BBB.


To validate the underlying concept of a conjugate consisting of a small brain imaging tracer and a large peptidic carrier molecule, a conjugate of the high affinity D2 receptor ligand fallypride and the TfR targeting peptide THRPPMWSPVWP has been synthesized. Furthermore, two derivatives of THRPPMWSPVWP were labeled with 68Ga in high radiochemical yields (>96%) and a radiochemical purity of 96–98% and evaluated in vitro and in vivo.


The fallypride–THRPPMWSPVWP conjugate still displayed a K i of 27 nM. The uptake of the 68Ga-labeled peptides into TfR-bearing cells was investigated using U87MG and HT-29 cells to assess the capability of the peptide to act as a carrier molecule targeting the TfR. The in vitro binding studies revealed negligible uptake of the tested 68Ga-labeled conjugates ranging from 0.08% to 0.66% after 60 min incubation at 37°C. Initial in vivo experiments with 68Ga-DOTA-S-maleimido–THRPPMWSPVWP in two healthy rats showed a mean brain uptake of 0.037% injected dose per gram, confirming the results obtained in vitro.


These results suggest that the accumulation of the 68Ga-radiolabeled conjugates of the TfR-binding peptide THRPPMWSPVWP into TFR expressing human cell lines is nonspecific and too low to render this peptide suitable as a possible carrier molecule for a receptor-mediated transport of compounds across the BBB.

Key words

Blood brain barrier (BBB) Active drug transport Transferrin receptor (TfR) 



This work was supported by research grants from the Natural Sciences and Engineering Council (NSERC) Canada and from the FRSQ funded Quebec Bio-Imaging Network to R. Schirrmacher.


  1. 1.
    Schreckenberger M, Hägele S, Siessmeier T, Buchholz H-G, Armbrust-Henrich H, Rösch F, Gründer G, Bartenstein P, Vogt T (2004) The dopamine D2 receptor ligand 18F–desmethoxyfallypride: an appropriate fluorinated PET tracer for the differential diagnosis of parkinsonism. Eur J Nucl Med Mol Imag 31(8):1128–1135CrossRefGoogle Scholar
  2. 2.
    Gründer G, Fellows C, Janouschek H, Veselinovic T, Boy C, Bröcheler A, Kirschbaum KM, Hellmann S, Spreckelmeyer KM, Hiemke C, Röch F, Schaefer WM, Vernaleken I (2008) Brain and plasma pharmacokinetics of aripiprazole in patients with schizophrenia: an [18F]Fallypride PET study. Am J Psychiatry 165:988–995PubMedCrossRefGoogle Scholar
  3. 3.
    Werhahn KJ, Landvogt C, Klimpe S, Buchholz HG, Yakushev I, Siessmeier T, Müller-Forell W, Piel M, Rösch F, Glaser M, Schreckenberger M, Bartenstein P (2006) Decreased dopamine D2/D3-receptor binding in temporal lobe epilepsy: an [18F]Fallypride PET study. Epilepsia 47(8):1392–1396PubMedCrossRefGoogle Scholar
  4. 4.
    Catafau AM, Suarez M, Bullich S, Llop J, Nucci G, Gunn RN, Brittain C, Laruelle M (2009) Within-subject comparison of striatal D2 receptor occupancy measurements using [123I]IBZM SPECT and [11C]Raclopride PET. Neuroimage 46:447–458PubMedCrossRefGoogle Scholar
  5. 5.
    Reeves S, Brown R, Howard R, Grasby P (2009) Increased striatal dopamine (D2/D3) receptor availability and delusions in Alzheimer disease. Neurology 72:528–534PubMedCrossRefGoogle Scholar
  6. 6.
    Odano I, Halldin C, Karlsson P, Varrone A, Airaksinen AJ, Krasikova RN, Farde L (2009) [18F]Flumazenil binding to central benzodiazepine receptor studies by PET—quantitative analysis and comparisons with [11C]flumazenil. Neuroimage 45:891–902PubMedCrossRefGoogle Scholar
  7. 7.
    Heiss W-D, Sobesky J (2008) Comparison of PET and DW/PW-MRI in acute ischemic stroke. Keio J Med 57(3):125–131PubMedCrossRefGoogle Scholar
  8. 8.
    Fisher PM, Meltzer CC, Price JC, Coleman RL, Ziolko SK, Becker C, Moses-Kolko EL, Berga SL, Hariri AR (2009) Medial prefrontal cortex 5-HT2A density is correlated with amygdala reactivity, response habituation, and functional coupling. Cereb Cortex 19(11):2499–2507PubMedCrossRefGoogle Scholar
  9. 9.
    Erritzoe D, Rasmussen H, Kristiansen KT, Frokjaer VG, Haugbol S, Pinborg L, Baaré W, Svarer C, Madsen J, Lublin H, Knudsen GM, Glenthoj BY (2008) Cortical and subcortical 5-HT2A receptor binding in neuroleptic-naive first-episode schizophrenic patients. Neuropsychopharmacology 33:2435–2441PubMedCrossRefGoogle Scholar
  10. 10.
    Cilia R, Marotta G, Benti R, Pezzoli G, Antonini A (2005) Brain SPECT imaging in multiple system atrophy. J Neural Transm 112:1635–1645PubMedCrossRefGoogle Scholar
  11. 11.
    Leenders KL (2003) Significance of non-presynaptic SPECT tracer methods in Parkinson’s disease. Mov Disord 18(suppl7):S39–S42PubMedCrossRefGoogle Scholar
  12. 12.
    Eshuis SA, Jager PL, Maguire RP, Jonkman S, Dierck RA, Leenders KL (2009) Direct comparison of FP-CIT SPECT and F-DOPA PET in patients with Parkinson’s disease and healthy controls. Eur J Nucl Med Mol Imag 36:454–462CrossRefGoogle Scholar
  13. 13.
    Qian ZM, Li H, Sun H, Ho K (2002) Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway. Pham Rev 54(4):561–587CrossRefGoogle Scholar
  14. 14.
    de Boer AG, Gaillard PJ (2007) Drug targeting to the brain. Ann Rev Pharmacol Toxicol 47:323–355CrossRefGoogle Scholar
  15. 15.
    Pardridge WM (2008) Re-engineering biopharmaceuticals for delivery to brain with molecular trojan horses. Bioconjug Chem 19(7):1327–1338PubMedCrossRefGoogle Scholar
  16. 16.
    Jones AR, Shusta EV (2007) Blood–brain barrier transport of therapeutics via receptor-mediation. Pharm Res 24(9):1759–1771PubMedCrossRefGoogle Scholar
  17. 17.
    Calzolari A, Oliviero I, Deaglio S, Mariani G, Biffoni M, Sposi NM, Malavasi F, Peschle C, Testa U (2007) Transferrin receptor 2 is frequently expressed in human cancer cell lines. Blood Cells Mol Dis 39:82–91PubMedCrossRefGoogle Scholar
  18. 18.
    Shin S-U, Friden P, Moran M, Olson T, Kang Y-S, Pardridge WM, Morrison SL (1995) Transferrin-antibody fusion proteins are effective in brain targeting. Proc Natl Acad Sci 92:2820–2824PubMedCrossRefGoogle Scholar
  19. 19.
    Mishera V, Mahor S, Rawat A, Gupta PN, Dubey P, Khatri K, Vyas SP (2006) Targeted brain delivery of AZT via transferrin anchored pegylated albumin nanoparticles. J Drug Target 14(1):45–53CrossRefGoogle Scholar
  20. 20.
    Ulbrich K, Hekmatara T, Herbert E, Kreuter J (2009) Transferrin- and transferrin-receptor-antibody-modified nanoparticles enable drug delivery across the blood–brain barrier (BBB). Eur J Pharm Biopharm 71:251–256PubMedCrossRefGoogle Scholar
  21. 21.
    Aktas Y, Yemisci M, Andrieux K, Gürsoy RN, Alonso MJ, Fernandez-Megia E, Novoa-Carballal R, Quinoa E, Riguera R, Sargon MF, Celik HH, Demir AS, Hıncal AA, Dalkara T, Capan Y, Couvreur P (2005) Development and brain delivery of chitosan-PEG nanoparticles functionalized with the monoclonal antibody OX26. Bioconj Chem 16:1503–1511CrossRefGoogle Scholar
  22. 22.
    Pang Z, Lu W, Gao H, Hu K, Chen J, Zhang C, Gao X, Jiang X, Zhu C (2008) Preparation and brain delivery property of biodegradable polymersomes conjugated with OX26. J Contr Rel 128:120–127CrossRefGoogle Scholar
  23. 23.
    Beduneau A, Saulnier P, Hindre F, Clavreul A, Leroux J-C, Benoit J-P (2007) Design of targeted lipid nanocapsules by conjugation of whole antibodies and antibody Fab’ fragments. Biomat 28:4978–4990CrossRefGoogle Scholar
  24. 24.
    Hackel BJ, Huang D, Bubolz JC, Wang XX, Shusta EV (2006) Production of soluble and active transferrin receptor-targeting single-chain antibody using Saccharomyces cerevisiae. Pharm Res 23(4):790–797PubMedCrossRefGoogle Scholar
  25. 25.
    Lee JH, Engler JA, Collawn JF, Moore BA (2001) Receptor mediated uptake of peptides that bind the human transferrin receptor. Eur J Biochem 268:2004–2012PubMedCrossRefGoogle Scholar
  26. 26.
    Oh S, Kim BJ, Singh NP, Lai H, Sasaki T (2009) Synthesis and anti-cancer activity if colvalent conjugates of artemisinin and a transferrin-receptor targeting peptide. Cancer Lett 274:33–39PubMedCrossRefGoogle Scholar
  27. 27.
    Moos T, Morgan EH (2001) Restricted transport of anti-transferrin receptor antibody (OX26) through the blood–brain barrier in the rat. J Neurochem 79(1):119–129PubMedCrossRefGoogle Scholar
  28. 28.
    Wängler C, Wängler B, Eisenhut M, Haberkorn U, Mier W (2008) Improved syntheses and applicability of different DOTA building blocks for multiply derivatized scaffolds. Bioorg Med Chem 16:2606–2616PubMedCrossRefGoogle Scholar
  29. 29.
    Wängler B, Quandt G, Iovkova L, Schirrmacher E, Wängler C, Boening G, Hacker M, Schmoeckel M, Jurkschat M, Bartenstein P, Schirrmacher R (2009) Kit-like 18F-labeling of proteins: synthesis of 4-(di-tert-butyl[18F]fluorosilyl)benzenethiol (Si[18F]FA-SH) labeled rat serum albumin for blood pool imaging with PET. Bioconj Chem 20:317–321CrossRefGoogle Scholar
  30. 30.
    Wellings DA, Atherton E (1997) Standard Fmoc protocols. Meth Enzymol 289:44–67PubMedCrossRefGoogle Scholar
  31. 31.
    Prante O, Einsiedel J, Haubner R, Gmeiner P, Wester HJ, Kuwert T, Maschauer S (2007) 3, 4, 6-Tri-O-acetyl-2-deoxy-2-[18F]fluoroglucopyranosyl phenylthio-sulfonate: a thiol-reactive agent for the chemoselective 18F-glycosylation of peptides. Bioconjug Chem 18(1):254–262PubMedCrossRefGoogle Scholar
  32. 32.
    Malmberg A, Jerning E, Mohell N (1996) Critical reevaluation of spiperone and benzamide binding to dopamine D2 receptors: evidence for identical binding sites. Eur J Pharmacol 303:123–128PubMedCrossRefGoogle Scholar
  33. 33.
    Schirrmacher R, Bradtmöller G, Schirrmacher E, Thews O, Tillmanns J, Siessmeier T, Buchholz HG, Bartenstein P, Wängler B, Niemeyer CM, Jurkschat K (2006) 18F-labeling of peptides by means of an organosilicon-based fluoride acceptor. Angew Chem Int Ed Engl 45(36):6047–6050PubMedCrossRefGoogle Scholar
  34. 34.
    Schirrmacher E, Wängler B, Cypryk M, Bradtmöller G, Schäfer M, Eisenhut M, Jurkschat K, Schirrmacher R (2007) Synthesis of p-(di-tert-butyl[18F]fluorosilyl)benzaldehyde ([18F]SiFA-A) with high specific activity by isotopic exchange: a convenient labeling synthon for the 18F-labeling of N-amino-oxy derivatized peptides. Bioconjug Chem 18(6):2085–2089PubMedCrossRefGoogle Scholar

Copyright information

© Academy of Molecular Imaging and Society for Molecular Imaging 2010

Authors and Affiliations

  • Carmen Wängler
    • 1
    • 2
    Email author
  • Dina Nada
    • 1
    • 2
  • Georg Höfner
    • 3
  • Simone Maschauer
    • 4
  • Björn Wängler
    • 5
  • Stephan Schneider
    • 6
  • Esther Schirrmacher
    • 1
  • Klaus T. Wanner
    • 3
  • Ralf Schirrmacher
    • 1
    • 2
    Email author
  • Olaf Prante
    • 4
  1. 1.McConnell Brain Imaging CentreMcGill UniversityMontrealCanada
  2. 2.Lady Davis Institute for Medical ResearchMcGill UniversityMontrealCanada
  3. 3.Pharmaceutical ChemistryLudwig-Maximilians-University MunichMunichGermany
  4. 4.Laboratory of Molecular Imaging, Clinic of Nuclear MedicineFriedrich-Alexander UniversityErlangenGermany
  5. 5.Department of Nuclear MedicineUniversity Hospital Munich, Ludwig-Maximilians University MunichMunichGermany
  6. 6.Department of Internal Medicine I, Division of Endocrinology and MetabolismBerufsgenossenschaftliches University Hospital Bergmannsheil, Ruhr-University BochumBochumGermany

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