Dissociation of 19F and fluorescence signal upon cellular uptake of dual-contrast perfluorocarbon nanoemulsions

  • Pascal Bouvain
  • Vera Flocke
  • Wolfgang Krämer
  • Rolf Schubert
  • Jürgen Schrader
  • Ulrich FlögelEmail author
  • Sebastian Temme
Research Article



Perfluorocarbon nanoemulsions (PFCs) tagged with fluorescence dyes have been intensively used to confirm the in vivo 19F magnetic resonance imaging (MRI) localization of PFCs by post mortem histology or flow cytometry. However, only limited data are available on tagged PFCs and the potential dissociation of fluorescence and 19F label after cellular uptake over time.

Materials and methods

PFCs were coupled to rhodamine (Rho) or carboxyfluorescein (Cfl) and their fate was analyzed after in vitro uptake by J774, RAW and CHO cells by flow cytometry and 19F MRI. In separate in vivo experiments, the dual-labelled emulsions were intravenously applied into mice and their distribution was monitored in spleen and liver over 24 h. In a final step, time course of fluorescence and 19F signals from injected emulsions were tracked in a local inflammation model making use of a subcutaneous matrigel depot doped with LPS (lipopolysaccharide).


Internalization of fluorescence-labelled PFCs was associated with a substantial whitening over 24 h in all macrophage cell lines while the 19F signal remained stable over time. In all experiments, CflPFCs were more susceptible to bleaching than RhoPFCs. After intravenous injection of RhoPFCs, the fluorescence signal in spleen and liver peaked after 30 min and 2 h, respectively, followed by a successive decrease over 24 h, whereas the 19F signal continuously increased during this observation period. Similar results were found in the matrigel/LPS model, where we observed increasing 19F signals in the inflammatory hot spot over time while the fluorescence signal of immune cells isolated from the matrigel depot 24 h after its implantation was only marginally elevated over background levels. This resulted in a massive underestimation of the true PFC deposition in the reticuloendothelial system and at inflammatory hot spots.


Cellular uptake of fluorescently tagged PFCs leads to a dissociation of the fluorescence and the 19F label signal over time, which critically impacts on interpretation of long-term experiments validated by histology or flow cytometry.


19F MRI Perfluorocarbons Inflammation Fluorescence Contrast media 



We like to thank Bodo Steckel and Sabine Barnert for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants ST 1209/1-1, FL 303/6-1 and the Sonderforschungsbereich SFB 1116.

Author contributions

Study conception and design: PB, JS, RS, ST, UF. Acquisition of data: PB, WK, VF, ST. Analysis and interpretation of data: PB, WK, VF, ST, UF. Drafting of manuscript: PB, ST, UF. Critical revision: UF, ST, JS, RS.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted.

Supplementary material

10334_2018_723_MOESM1_ESM.tif (8.6 mb)
Supplementary material 1 Chemical structure of fluorescent lipids used for the generation of CflPFCs [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein)] and RhoPFCs [Lissamine™ Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine]. (TIFF 8795 kb)


  1. 1.
    Bulte JWM (2005) Hot spot MRI emerges from the background. Nat Biotechnol 23:945–946CrossRefPubMedGoogle Scholar
  2. 2.
    Riess JG (2005) Understanding the fundamentals of Perfluorocarbons and perfluorocarbon emulsions relevant to in vivo oxygen delivery. Artif Cells Blood Substit Biotechnol 33:47–63CrossRefGoogle Scholar
  3. 3.
    O’Hagan D (2008) Understanding organofluorine chemistry. An introduction to the C–F bond. Chem Soc Rev 37:308–319CrossRefPubMedGoogle Scholar
  4. 4.
    Grapentin C, Mayenfels F, Barnert S, Süss R, Schubert R, Temme S, Jacoby C, Schrader J, Flögel U (2014) Optimization of perfluorocarbon nanoemulsions for molecular imaging by 19F MRI. Nanomed One Central Press, Machester, pp 268–286Google Scholar
  5. 5.
    Srinivas M, Boehm-Sturm P, Figdor CG, de Vries IJ, Hoehn M (2012) Labeling cells for in vivo tracking using 19F MRI. Biomaterials 33:8830–8840CrossRefPubMedGoogle Scholar
  6. 6.
    Temme S, Grapentin C, Güden-Silber T, Flögel U (2016) Active targeting of perfluorocarbon nanoemulsions. Fluorine magnetic resonance imaging. CRC Press, Boca Raton, pp 97–133Google Scholar
  7. 7.
    Ebner B, Behm P, Jacoby C, Burghoff S, French BA, Schrader J, Flögel U (2010) Early assessment of pulmonary inflammation by 19F MRI in vivo. Circ Cardiovasc Imaging 3:202–210CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Flögel U, Ding Z, Hardung H, Jander S, Reichmann G, Jacoby C, Schubert R, Schrader J (2008) In vivo monitoring of inflammation after cardiac and cerebral ischemia by fluorine magnetic resonance imaging. Circulation 118:140–148CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Flögel U, Burghoff S, van Lent PLEM, Temme S, Galbarz L, Ding Z, El-Tayeb A, Huels S, Bönner F, Borg N, Jacoby C, Muller CE, van den Berg WB, Schrader J (2012) Selective activation of adenosine A2A receptors on immune cells by a CD73-dependent prodrug suppresses joint inflammation in experimental rheumatoid arthritis. Sci Transl Med 4:146ra108CrossRefPubMedGoogle Scholar
  10. 10.
    van Heeswijk RB, Pellegrin M, Flögel U, Gonzales C, Aubert J-F, Mazzolai L, Schwitter J, Stuber M (2015) Fluorine MR imaging of inflammation in atherosclerotic plaque in vivo. Radiology 275:421–429CrossRefPubMedGoogle Scholar
  11. 11.
    Boehm-Sturm P, Mengler L, Wecker S, Hoehn M, Kallur T (2011) In vivo tracking of human neural stem cells with 19F magnetic resonance imaging. PLoS One 6:e29040CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Helfer BM, Balducci A, Sadeghi Z, Ohanlon C, Hijaz A, Flask CA, Wesa A (2013) 19F MRI tracer preserves in vitro and in vivo properties of hematopoietic stem cells. Cell Transplant 22:87–97CrossRefPubMedGoogle Scholar
  13. 13.
    Ding Z, Temme S, Quast C, Friebe D, Jacoby C, Zanger K, Bidmon H-J, Grapentin C, Schubert R, Flogel U, Schrader J (2016) Epicardium-derived cells formed after myocardial injury display phagocytic activity permitting in vivo labeling and tracking. Stem Cells Transl Med 5:639–650CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Solomon M, Muro S (2017) Lysosomal enzyme replacement therapies: historical development, clinical outcomes, and future perspectives. Adv Drug Deliv Rev 118:109–134CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Dupré-Crochet S, Erard M, Nüße O (2013) ROS production in phagocytes: why, when, and where? J Leukoc Biol 94:657–670CrossRefPubMedGoogle Scholar
  16. 16.
    Temme S, Jacoby C, Ding Z, Bönner F, Borg N, Schrader J, Flögel U (2014) Technical advance: monitoring the trafficking of neutrophil granulocytes and monocytes during the course of tissue inflammation by noninvasive 19F MRI. J Leukoc Biol 95:689–697CrossRefPubMedGoogle Scholar
  17. 17.
    Temme S, Grapentin C, Quast C, Jacoby C, Grandoch M, Ding Z, Owenier C, Mayenfels F, Fischer JW, Schubert R, Schrader J, Flögel U (2015) Noninvasive imaging of early venous thrombosis by 19F magnetic resonance imaging with targeted perfluorocarbon nanoemulsions. Circulation 131:1405–1414CrossRefPubMedGoogle Scholar
  18. 18.
    Vasir JK, Labhasetwar V (2007) Biodegradable nanoparticles for cytosolic delivery of therapeutics. Adv Drug Deliv Rev 59:718–728CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Skotland T, Sontum PC, Oulie I (2002) In vitro stability analyses as a model for metabolism of ferromagnetic particles (Clariscan™), a contrast agent for magnetic resonance imaging. J Pharm Biomed Anal 28:323–329CrossRefPubMedGoogle Scholar
  20. 20.
    Seleverstov O, Zabirnyk O, Zscharnack M, Bulavina L, Nowicki M, Heinrich J-M, Yezhelyev M, Emmrich F, O’Regan R, Bader A (2006) Quantum dots for human mesenchymal stem cells labeling. A size-dependent autophagy activation. Nano Lett 6:2826–2832CrossRefPubMedGoogle Scholar
  21. 21.
    Fitzpatrick JAJ, Andreko SK, Ernst LA, Waggoner AS, Ballou B, Bruchez MP (2009) Long-term persistence and spectral blue shifting of quantum dots in vivo. Nano Lett 9:2736–2741CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Ratner AV, Hurd R, Muller HH, Bradley-Simpson B, Pitts W, Shibata D, Sotak C, Young SW (1987) 19F magnetic resonance imaging of the reticuloendothelial system. Magn Reson Med 5:548–554CrossRefPubMedGoogle Scholar
  23. 23.
    Long DM, Multer FK, Greenburg AG, Peskin GW, Lasser EC, Wickham WG, Sharts CM (1978) Tumor imaging with X-rays using macrophage uptake of radiopaque fluorocarbon emulsions. Surgery 84:104–112PubMedGoogle Scholar
  24. 24.
    Jacoby C, Temme S, Mayenfels F, Benoit N, Krafft MP, Schubert R, Schrader J, Flögel U (2014) Probing different perfluorocarbons for in vivo inflammation imaging by 19F MRI: image reconstruction, biological half-lives and sensitivity. NMR Biomed 27:261–271CrossRefPubMedGoogle Scholar
  25. 25.
    Flaim SF (1994) Pharmacokinetics and side effects of perfluorocarbon-based blood substitutes. Artif Cells Blood Substit Immobil Biotechnol 22:1043–1054CrossRefPubMedGoogle Scholar
  26. 26.
    Krafft MP, Riess JG, Weers JG (1998) The design and engineering of oxygen-delivering fluorocarbonemulsions. In: Benita S (ed) Submicron emulsions in drug targeting and delivery. Harwood, Amsterdam, pp 235–333Google Scholar
  27. 27.
    Riess JG (2001) Oxygen carriers (“blood substitutes”)–raison d’etre, chemistry, and some physiology. Chem Rev 101:2797–2920CrossRefPubMedGoogle Scholar
  28. 28.
    Tsuda Y, Yamanouchi K, Yokoyama K, Suyama T, Watanabe M, Ohyanagi H, Saitoh Y (1988) Discussion and considerations for the excretion mechanism of perfluorochemical emulsion. Biomater Artif Cells Artif Organs 16:473–483CrossRefPubMedGoogle Scholar
  29. 29.
    Yokoyama K, Yamanouchi K, Murashima R (1975) Excretion of Perfluorochemicals after intravenous injection of their emulsion. Chem Pharm Bull (Tokyo) 23:1368–1373CrossRefGoogle Scholar
  30. 30.
    Riess JG (1992) Overview of progress in the fluorocarbon approach to in vivo oxygen delivery. Biomater Artif Cells Immobil Biotechnol 20:183–202Google Scholar
  31. 31.
    Bönner F, Jacoby C, Temme S, Borg N, Ding Z, Schrader J, Flögel U (2014) Multifunctional MR monitoring of the healing process after myocardial infarction. Basic Res Cardiol 109:430CrossRefPubMedGoogle Scholar
  32. 32.
    Skajaa T, Zhao Y, van den Heuvel DJ, Gerritsen HC, Cormode DP, Koole R, van Schooneveld MM, Post JA, Fisher EA, Fayad ZA, de Mello Donega C, Meijerink A, Mulder WJM (2010) Quantum dot and Cy5.5 labeled nanoparticles to investigate lipoprotein biointeractions via Förster resonance energy transfer. Nano Lett 10:5131–5138CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Lunov O, Syrovets T, Röcker C, Tron K, Ulrich Nienhaus G, Rasche V, Mailänder V, Landfester K, Simmet T (2010) Lysosomal degradation of the carboxydextran shell of coated superparamagnetic iron oxide nanoparticles and the fate of professional phagocytes. Biomaterials 31:9015–9022CrossRefPubMedGoogle Scholar
  34. 34.
    Gálisová A, Herynek V, Swider E, Sticová E, Pátiková A, Kosinová L, Kříž J, Hájek M, Srinivas M, Jirák D (2018) A trimodal imaging platform for tracking viable transplanted pancreatic islets in vivo: F-19 MR, fluorescence, and bioluminescence imaging. Mol Imaging Biol. CrossRefPubMedCentralGoogle Scholar
  35. 35.
    Janjic JM, Srinivas M, Kadayakkara DKK, Ahrens ET (2008) Self-delivering nanoemulsions for dual fluorine-19 MRI and fluorescence detection. J Am Chem Soc 130:2832–2841CrossRefPubMedGoogle Scholar

Copyright information

© European Society for Magnetic Resonance in Medicine and Biology (ESMRMB) 2018

Authors and Affiliations

  1. 1.Experimental Cardiovascular Imaging, Department of Molecular CardiologyHeinrich Heine University of DüsseldorfDüsseldorfGermany
  2. 2.Department of Engineering PhysicsUniversity of Applied Science MünsterMünsterGermany
  3. 3.Pharmaceutical Technology and BiopharmacyAlbert Ludwigs UniversityFreiburgGermany

Personalised recommendations