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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 19 F-combined with 1 H-MRI. In vivo and ex vivo 1 H/ 19 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. Results: After injection of EP9-PFC-NE, the in-vivo 19 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. 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., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)