Abstract 1401 Specificity and translational potential of imaging MI-activated cardiac stromal cells in-vivo by 19F-MRI.

Introduction: Magnetic resonance imaging (¹HMRI) is a noninvasive tool for cardiac visualization with high spatial resolution and contrast with soft tissues. Targeted ¹⁹F MRI permits the background-free assessment of binding to specific cell types in the heart. After MI, epicardium-derived stromal cells (EpiSCs) become activated and differentiate into various cardiac cell types that support the healing process. Cardiac fibroblasts (CFs) become activated contributing to scar formation, protecting the structural integrity of the infarcted heart. However, prolonged activation can promote adverse remodeling. A non-invasive targeting method for imaging activated cardiac stromal cells would be advantageous because of their importance in cardiac repair, and as a potential therapeutic target.

Methods: Targeting was achieved by using peptides recently identified by our group (Straub et al.,2020), which specifically bind to EpiSCs. The specificity of five peptides (EP1-EP5) was tested in vitro in EpiSCs and CFs isolated from the infarcted mouse heart. Flow cytometric analysis was performed using a BD FACS Canto II. To confirm the specificity of targeting, scrambled and mutated peptides were used as negative controls. We also studied binding to human cells: monocytes, dermal fibroblasts, and CFs isolated from surgical specimens. For in-vivo imaging, we used a delivery system based on nanoemulsions (NEs) composed of a mixture of phospholipids coated with polyethylene glycol functionalized with the targeting peptide.To allow imaging, the system was loaded with perfluorocarbons (PFCs) for visualization by 19F-MRI with ¹H-MRI. PFC-NEs were generated by high-pressure homogenization. In vivo and ex vivo ¹H/¹⁹F MRI was performed on a Bruker 9.4 T AVANCE III wide-bore NMR spectrometer. Specific identification of the cell types that incorporate PFC-NEs was done by transmission electron microscopy (TEM).

Results: The previously characterized peptide (EP5) was tested in several mouse cells showing a high binding capacity in EpiSC and CFs. EP5 was therefore incorporated into the NEs as described above. In vivo targeting of peptide-decored liposomes to cardiac stromal cells was done in a mouse MI model (50 minutes ischemia/reperfusion). NEs were injected via tail vein 5 days after the induction of MI. The ability to target the infarcted heart (EpiSC and CFs) was evaluated by 1H/19F-MRI 24 hours after injection using high-resolution in-vivo and ex-vivo visualization of the heart. The 19F signal was localized within the infarcted area and was EP5-specific since mutation of the peptide significantly lowered the signal.Temporal in-vivo analysis of blood after injection revealed that NEs have a long circulation time (>24h). The stromal cell-specific uptake in the post-MI heart was verified by TEM: EP5-NEs were found to accumulate within cells of the epicardial layer and CFs, but not in macrophages and cardiomyocytes. Since EP5 is also strongly bound to human CFs but not to human monocytes, our approach may apply to the human situation.

Conclusions: Our findings show that peptide targeting of NEs is specific for cardiac stromal cells in mice and men, and permits the in vivo imaging of these cells in the post-MI mouse heart. This nano-based system has translational potential and also appears to be suitable for the specific delivery of therapeutic compounds to promote cardiac healing.

Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w
Specificity and translational potential of imaging MI-activated cardiac stromal cells in-vivo by 19F-MRI.
A. A. Euan Martinez¹, U. Flögel¹, Z. Ding¹, S. Temme¹, J. Schrader¹
¹Institut für Molekulare Kardiologie, Universitätsklinikum Düsseldorf, Düsseldorf;
Introduction: Magnetic resonance imaging (¹HMRI) is a noninvasive tool for cardiac visualization with high spatial resolution and contrast with soft tissues. Targeted ¹⁹F MRI permits the background-free assessment of binding to specific cell types in the heart. After MI, epicardium-derived stromal cells (EpiSCs) become activated and differentiate into various cardiac cell types that support the healing process. Cardiac fibroblasts (CFs) become activated contributing to scar formation, protecting the structural integrity of the infarcted heart. However, prolonged activation can promote adverse remodeling. A non-invasive targeting method for imaging activated cardiac stromal cells would be advantageous because of their importance in cardiac repair, and as a potential therapeutic target. Methods: Targeting was achieved by using peptides recently identified by our group (Straub et al.,2020), which specifically bind to EpiSCs. The specificity of five peptides (EP1-EP5) was tested in vitro in EpiSCs and CFs isolated from the infarcted mouse heart. Flow cytometric analysis was performed using a BD FACS Canto II. To confirm the specificity of targeting, scrambled and mutated peptides were used as negative controls. We also studied binding to human cells: monocytes, dermal fibroblasts, and CFs isolated from surgical specimens. For in-vivo imaging, we used a delivery system based on nanoemulsions (NEs) composed of a mixture of phospholipids coated with polyethylene glycol functionalized with the targeting peptide.To allow imaging, the system was loaded with perfluorocarbons (PFCs) for visualization by 19F-MRI with ¹H-MRI. PFC-NEs were generated by high-pressure homogenization. In vivo and ex vivo ¹H/¹⁹F MRI was performed on a Bruker 9.4 T AVANCE III wide-bore NMR spectrometer. Specific identification of the cell types that incorporate PFC-NEs was done by transmission electron microscopy (TEM). Results: The previously characterized peptide (EP5) was tested in several mouse cells showing a high binding capacity in EpiSC and CFs. EP5 was therefore incorporated into the NEs as described above. In vivo targeting of peptide-decored liposomes to cardiac stromal cells was done in a mouse MI model (50 minutes ischemia/reperfusion). NEs were injected via tail vein 5 days after the induction of MI. The ability to target the infarcted heart (EpiSC and CFs) was evaluated by 1H/19F-MRI 24 hours after injection using high-resolution in-vivo and ex-vivo visualization of the heart. The 19F signal was localized within the infarcted area and was EP5-specific since mutation of the peptide significantly lowered the signal.Temporal in-vivo analysis of blood after injection revealed that NEs have a long circulation time (>24h). The stromal cell-specific uptake in the post-MI heart was verified by TEM: EP5-NEs were found to accumulate within cells of the epicardial layer and CFs, but not in macrophages and cardiomyocytes. Since EP5 is also strongly bound to human CFs but not to human monocytes, our approach may apply to the human situation. Conclusions: Our findings show that peptide targeting of NEs is specific for cardiac stromal cells in mice and men, and permits the in vivo imaging of these cells in the post-MI mouse heart. This nano-based system has translational potential and also appears to be suitable for the specific delivery of therapeutic compounds to promote cardiac healing.
https://dgk.org/kongress_programme/jt2023/aV1994.html