Key result
EP9-PFC-NE selectively targets post-MI cardiac fibroblasts via CD63, enabling in vivo 19F MRI imaging.
CD63 is a novel target that triggers endocytosis of EP9-PFC-NE in activated cardiac fibroblasts, enabling in vivo imaging and potential targeted drug delivery.
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.
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Tellkamp et al. (2024) studied Myocardial infarction and cardiac fibrosis. EP9-PFC-NE (EP9-decorated perfluorocarbon-containing nanoemulsion) vs. Mutated peptide was evaluated on In vivo localization and cellular uptake. EP9-PFC-NE specifically targeted activated cardiac fibroblasts in the infarcted heart via binding to the tetraspanin CD63, enabling in vivo 19F MRI imaging and cellular uptake.
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