CD63 as novel target for nanoemulsion-based F MRI imaging and drug delivery to activated cardiac fibroblasts.
Arlen Aurora Euan Martínez, Ann Kathrin Bergmann, Frederik Tellkamp, Stephan Schott-Verdugo, Pascal Bouvain, Julia Steinhausen, Jasmin Bahr, Vivien Kmietczyk, Maja Bencun, Ulrich Flögel, Jörg H W Distler, Marcus Krueger, Mirko Völkers, Constantin Czekelius, Holger Gohlke, Sebastian Temme, Julia Hesse, Jürgen Schrader
Author Information
Arlen Aurora Euan Martínez: Department of Molecular Cardiology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Ann Kathrin Bergmann: Core Facility for Electron Microscopy, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Frederik Tellkamp: Institute for Genetics, University of Cologne, Cologne, Germany.
Stephan Schott-Verdugo: Institute of Bio- and Geosciences (IBG-4: Bioinformatics), Forschungszentrum Jülich, Jülich, Germany.
Pascal Bouvain: Department of Molecular Cardiology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Julia Steinhausen: Department of Molecular Cardiology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Jasmin Bahr: Department of Molecular Cardiology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Vivien Kmietczyk: Department of Internal Medicine III (Cardiology, Angiology, and Pneumology), Heidelberg University Hospital, Heidelberg, Germany.
Maja Bencun: Department of Internal Medicine III (Cardiology, Angiology, and Pneumology), Heidelberg University Hospital, Heidelberg, Germany.
Ulrich Flögel: Department of Molecular Cardiology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Jörg H W Distler: Department of Rheumatology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Marcus Krueger: Institute for Genetics, University of Cologne, Cologne, Germany.
Mirko Völkers: Department of Internal Medicine III (Cardiology, Angiology, and Pneumology), Heidelberg University Hospital, Heidelberg, Germany.
Constantin Czekelius: Institute for Organic Chemistry and Macromolecular Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Holger Gohlke: Institute of Bio- and Geosciences (IBG-4: Bioinformatics), Forschungszentrum Jülich, Jülich, Germany.
Sebastian Temme: CARID, Cardiovascular Research Institute Düsseldorf, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Julia Hesse: Department of Molecular Cardiology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Jürgen Schrader: Department of Molecular Cardiology, Medical Faculty and University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
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. Targeting was achieved by linking a 7-amino acid peptide (EP9) to a perfluorocarbon-containing nanoemulsion (PFC-NE) for visualization by F-combined with H-MRI. and H/F 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. After injection of EP9-PFC-NE, the in-vivo F 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. 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 imaging and has the potential for fibroblast-specific drug delivery.
