A Langendorff-heart discovery pipeline demonstrates cardiomyocyte targeting by extracellular vesicles functionalized...

Extracellular vesicles (EVs) are widely explored as vehicles for delivering therapeutic or experimental cargo to cardiomyocytes. Efforts to improve EV bioavailability in the heart, and reduce their off-target actions, require screening methods that can replicate the physiological and anatomical barriers present in the myocardium. Additionally, discovery pipelines must exercise control over EV dosage and timing, and provide a means of assessing cargo incorporation into cardiomyocytes specifically. These criteria are not generally met by experiments on cultured cells or animals. Here, we present a Langendorff-heart discovery pipeline that combines the strengths of in vivo and in vitro approaches. Langendorff-mode perfusion enables controlled exposure of beating hearts to re-circulated EVs. Following perfusion, cardiomyocytes can be isolated enzymatically for analysis, such as imaging. We tested this discovery pipeline by functionalizing EVs with beta-blockers (atenolol, metoprolol) using click-chemistry and incorporating the fluorescent protein NeonGreen2 to track the fate of EV cargo. Fluorescence in cardiomyocytes, including their nuclear regions, increased after Langendorff-treatment with beta-blocker decorated EVs, but only if these contained NeonGreen2, implicating the fluorescent cargo as the source of signal. Superior binding efficacy of beta-blockers was confirmed by referencing to the substantially lower signals obtained using wild-type EVs or EVs presenting myomaker or myomixer proteins, motifs that modestly enrich cardiac EV uptake in mice. Our findings demonstrate successful cardiomyocyte targeting using EVs decorated with beta-receptor binders. We propose the Langendorff-perfused heart as an intermediate step - nested between in vitro characterisation and animal testing - in discovery pipelines for seeking improved cardiac-specific EV designs.