Abstract 11467: Biomimetic Microrna-Laden Small Extracellular Vesicle-Derived Vehicles Increase Pro-Reparative Phenotype in Cardiac Endothelial Cells

Abstract

Introduction: Ckit+ progenitor cells (CPCs) release small extracellular vesicles (sEVs) which are vital for CPC-based recovery after myocardial infarction (MI). sEVs transport RNA cargo within a complex lipid/protein bilayer required for sEV trafficking and endocytosis. However, being cell-derived, sEV yield, cargo content and concentration are often variable. Synthetic sEV-mimics allow for cargo control, but their basic phospholipid membrane often results in rapid clearance. Here, we use thin film hydration (TFH) and electroporation to develop sEV-like vesicles (ELVs) with an sEV membrane and mimic-like cargo control. We hypothesize that (1) ELVs with cardioprotective microRNA (miR) cargo will improve functional outcomes from cardiac endothelial cells (CECs) compared to sEVs alone and (2) electroporated ELVs will functionally outperform TFH ELVs. Methods: sEVs were isolated from CPC conditioned media with ultracentrifugation. ELVs were synthesized from sEVs by removing inherent miR cargo and then loading endothelial specific miR126 through TFH or electroporation. For TFH, sEVs were dissolved in an organic solvent to form a lipid bilayer and then loaded with miR126 to initiate ELV self-assembly. For electroporation, miR126 was added to sEVs by varying voltage pulses. ELVs were characterized for size and concentration. Both cargo clearance and miR126 loading were quantified with a Quant-iT miR kit. Functionality of both ELV designs was assessed by delivering miR126+ ELVs to CECs for angiogenesis and proliferation. Results: ELVs on a similar scale to sEVs were successfully formed using both methods, despite variability in concentration. ELV miR126 loading capacity after cargo removal was higher and tunable with electroporation compared to TFH. Electroporation also preserved more membrane proteins than TFH. When delivered to CECs, electroporated miR126+ ELVs increased angiogenesis and proliferation compared to sEVs over 24 hours, in a dose-dependent manner. Conclusion: Using an sEV-like membrane with customizable miR cargo allows for improved functional outcomes from cardiac cells, with electroporated samples having more cargo tunability. Beyond miR126, this work highlights a delivery vehicle for combination miR therapy after MI.