CD63 as novel target for nanoemulsion-based 19F MRI imaging and drug delivery to activated cardiac fibroblasts

  • Theranostics. 2025 Jan 1;15(1):1-18. doi: 10.7150/thno.96990.
Arlen Aurora Euan Martínez  1  2 ,  Ann Kathrin Bergmann  3 ,  Frederik Tellkamp  4  5 ,  Stephan Schott-Verdugo  6 ,  Pascal Bouvain  1 ,  Julia Steinhausen  1 ,  Jasmin Bahr  1 ,  Vivien Kmietczyk  7  8 ,  Maja Bencun  7  8 ,  Ulrich Flögel  1  9 ,  Jörg H W Distler  10  11 ,  Marcus Krueger  4  5 ,  Mirko Völkers  7  8 ,  Constantin Czekelius  12 ,  Holger Gohlke  6  13 ,  Sebastian Temme  9  14 ,  Julia Hesse  1  9 ,  Jürgen Schrader  1  9
Affiliations
  • 1. Department of Molecular Cardiology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
  • 2. Crozet-Medical GmbH, Düsseldorf, Germany.
  • 3. Core Facility for Electron Microscopy, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
  • 4. Institute for Genetics, University of Cologne, Cologne, Germany.
  • 5. Cologne Excellence Cluster on Stress Responses in Aging-associated Diseases (CECAD), University of Cologne, Cologne, Germany.
  • 6. Institute of Bio- and Geosciences (IBG-4: Bioinformatics), Forschungszentrum Jülich, Jülich, Germany.
  • 7. Department of Internal Medicine III (Cardiology, Angiology, and Pneumology), Heidelberg University Hospital, Heidelberg, Germany.
  • 8. DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim, Heidelberg, Germany.
  • 9. CARID, Cardiovascular Research Institute Düsseldorf, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
  • 10. Department of Rheumatology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
  • 11. Hiller Research Center, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
  • 12. Institute for Organic Chemistry and Macromolecular Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
  • 13. Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
  • 14. Department of Anesthesiology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
PMID: 39744226 DOI: 10.7150/thno.96990
Abstract

Rationale: Cardiac fibroblasts are activated following myocardial infarction (MI) and cardiac fibrosis is a major driver of the growing burden of heart failure. A non-invasive targeting method for activated cardiac fibroblasts would be advantageous because of their importance for imaging and therapy. Methods: Targeting was achieved by linking a 7-amino acid peptide (EP9) to a perfluorocarbon-containing nanoemulsion (PFC-NE) for visualization by 19F-combined with 1H-MRI. In vivo and ex vivo 1H/19F MRI was performed on a Bruker 9.4 T AVANCE III wide-bore nuclear magnetic resonance spectrometer. Photoaffinity labeling (diazirine photolinker) and mass spectrometry were used to identify the peptide-binding protein. Molecular modeling studies used ColabFold and AlphaFold 3. EP9-decorated liposomes containing modified mRNA for luciferase (mRNA-LUC) were used for the study of the cellular uptake process. Results: After injection of EP9-PFC-NE, the in-vivo 19F signal localized to the infarcted area of the heart and was EP9-specific, as verified by the use of a mutated peptide. The plasma half-life of the nanoemulsion was 20 h and electron microscopy identified cardiac fibroblasts and epicardial stromal cells to be the main populations for cellular uptake. Photoaffinity labeling identified the tetraspanin CD63 as the main EP9-binding protein, which was supported by CD63-EP9 modeling data. Expression of CD63 was significantly upregulated in infarct-activated fibroblasts of mice and humans. Cellular uptake may involve caveolae and/or clathrin-coated pits as suggested by scRNAseq data. Uptake studies with mRNA-LUC-loaded EP9-PFC-NE confirmed internalization after binding to fibroblast CD63. Conclusions: CD63 was identified to contain a specific EP9 binding motive that triggers endocytosis of EP9-PFC-NE in activated cardiac fibroblasts. This targeted nanoemulsion can therefore be used for in vivo imaging and has the potential for fibroblast-specific drug delivery.

Keywords
CD63; FAP; fibrosis; modified mRNA; myocardial infarction.
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