Abstract 497 In vivo imaging of epicardium-derived stromal cells formed after MI by 19F-MRI using a peptide targeted nanoemulsion

Introduction: During MI, the heart activates the proliferation of epicardium-derived stromal cells (EpiSC) which recapitulate an embryonic program. During cardiac development EpiSC still can differentiate into cardiomyocytes, fibroblasts, and endothelial cells. In the adult heart, EpiSC have retained a cardiogenic potential and can form cardiomyocytes after intravenous application of thymosin β4 (Smart et al. Nature. 2011). Systemic application of cardioactive drugs have the disadvantage of being rapidly degraded, so that their local concentration may be too low to be fully biologically active in the heart. To overcome these limitations, the use of a nano-based delivery system allows the encapsulation of drugs and contrast agents to permit the targeted delivery to specific cellular sites (theranostic).

Methods:Targeting was achieved by the use of a peptide recently identified by our group (Straub et al. 2020), which specifically binds to EpiSC. The specificity of five EpiSC specific peptides (EP1-EP5) was tested in vitro in the following cell-cultured lines: mice EpiSCs, mice cardiac fibroblasts, normal human dermal fibroblasts (NHDF), and human monocytes (THP-1). Flow cytometric analysis was performed, after two washing steps with MACS and staining with DAPI using a BD FACS Canto II. To further confirm the specific targeting of the selected peptide sequence, scrambled and mutated peptides were used as negative controls. Subsequently, the specific peptide was incorporated into a delivery system based on nanoemulsions (NEs) composed of a mixture of phospholipids and coated with polyethylene glycol that was functionalized with the targeting peptide. To allow imaging, the system was loaded with perfluorocarbons (PFCs) for visualization by 19F-MRI combined with 1H-MRI. PFC-NEs were generated by high-pressure homogenization at 1000 bar, using an LV1 microfluidizer. 1H / 19F MRI was performed on a Bruker 9.4 T AVANCE III wide-bore NMR spectrometer (Bruker BioSpin MRI GmbH, Ettlingen, Germany).

Results:Five previously characterized peptides (EP 1-5) were tested in several cell lines (listed above). These in vitro experiments identified EP5 as having the highest binding capacity. EP5 has the peptide sequence KLMLPRP and was incorporated into the NEs as described above. In vivo targeting of peptides to the heart was done in a mouse MI model (50 minutes ischemia / reperfusion). The NEs were applied via tail vein injection (250 µL) 5 days after the induction of MI. The abilty to target the heart in particular the EpiSC was evaluated by 1H / 19F-MRI in vivo 24 hours after injection, and high-resolution ex vivo visualization of the heart was carried out thereafter. Within the post MI heart we observed a strong signal of 19F located within the epicardial cell layer of the infarcted heart,

Conclusion: Our findings show that peptide targeting of NEs permits the in vivo imaging of EpiSC in the postMI heart. This nano-based system appears to be suitable for the specific delivery of therapeutic compounds eg thymosin ß4, to potentially promote cardiac healing.

Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5
In vivo imaging of epicardium-derived stromal cells formed after MI by 19F-MRI using a peptide targeted nanoemulsion
A. A. Euan Martinez¹, S. Temme¹, U. Flögel¹, C. Alter¹, Z. Ding¹, J. Schrader¹
¹Institut für Molekulare Kardiologie, Universitätsklinikum Düsseldorf, Düsseldorf;
Introduction: During MI, the heart activates the proliferation of epicardium-derived stromal cells (EpiSC) which recapitulate an embryonic program. During cardiac development EpiSC still can differentiate into cardiomyocytes, fibroblasts, and endothelial cells. In the adult heart, EpiSC have retained a cardiogenic potential and can form cardiomyocytes after intravenous application of thymosin β4 (Smart et al. Nature. 2011). Systemic application of cardioactive drugs have the disadvantage of being rapidly degraded, so that their local concentration may be too low to be fully biologically active in the heart. To overcome these limitations, the use of a nano-based delivery system allows the encapsulation of drugs and contrast agents to permit the targeted delivery to specific cellular sites (theranostic). Methods:Targeting was achieved by the use of a peptide recently identified by our group (Straub et al. 2020), which specifically binds to EpiSC. The specificity of five EpiSC specific peptides (EP1-EP5) was tested in vitro in the following cell-cultured lines: mice EpiSCs, mice cardiac fibroblasts, normal human dermal fibroblasts (NHDF), and human monocytes (THP-1). Flow cytometric analysis was performed, after two washing steps with MACS and staining with DAPI using a BD FACS Canto II. To further confirm the specific targeting of the selected peptide sequence, scrambled and mutated peptides were used as negative controls. Subsequently, the specific peptide was incorporated into a delivery system based on nanoemulsions (NEs) composed of a mixture of phospholipids and coated with polyethylene glycol that was functionalized with the targeting peptide. To allow imaging, the system was loaded with perfluorocarbons (PFCs) for visualization by 19F-MRI combined with 1H-MRI. PFC-NEs were generated by high-pressure homogenization at 1000 bar, using an LV1 microfluidizer. 1H / 19F MRI was performed on a Bruker 9.4 T AVANCE III wide-bore NMR spectrometer (Bruker BioSpin MRI GmbH, Ettlingen, Germany). Results:Five previously characterized peptides (EP 1-5) were tested in several cell lines (listed above). These in vitro experiments identified EP5 as having the highest binding capacity. EP5 has the peptide sequence KLMLPRP and was incorporated into the NEs as described above. In vivo targeting of peptides to the heart was done in a mouse MI model (50 minutes ischemia / reperfusion). The NEs were applied via tail vein injection (250 µL) 5 days after the induction of MI. The abilty to target the heart in particular the EpiSC was evaluated by 1H / 19F-MRI in vivo 24 hours after injection, and high-resolution ex vivo visualization of the heart was carried out thereafter. Within the post MI heart we observed a strong signal of 19F located within the epicardial cell layer of the infarcted heart, Conclusion: Our findings show that peptide targeting of NEs permits the in vivo imaging of EpiSC in the postMI heart. This nano-based system appears to be suitable for the specific delivery of therapeutic compounds eg thymosin ß4, to potentially promote cardiac healing.
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