Abstract 11898: Bovine Milk-Derived Extracellular Vesicles as a Novel Injury-Targeting Drug Delivery System Following Cardiac Injury

Abstract

Introduction: A novel protocol provides large amounts of highly purified small extracellular vesicles (also called exosomes) from bovine milk (Marsh et al, PMID: 34367882). We sought to examine the targeting potential of these pure, highly concentrated bovine milk-derived extracellular vesicles (mEVs) to injured cells and tissues, with the intent of identifying an effective drug delivery platform capable of targeting injured cardiac tissue. Methods: Targeting of mEVs to injured cells and tissues was tested in vitro using a scratch assay on human dermal fibroblast (hDFs) and MDCK cell monolayers, and in vivo, using mouse models of skin wounding and cardiac injury - methods we have previously reported (PMID:34246197; PMID:29351451). mEVs were isolated using our published approach (PMID: 34367882), then fluorescently tagged with Cell Tracker Deep Red (CTDR). Labelled mEVs were then applied to cell cultures at 20 ug/mL for 15 minutes post wound; cells were then rinsed, fixed and stained for cell nuclei with Hoechst. Mice were provided 2 ug/kg loaded mEV’s by oral gavage before skin surgery and induction of cardiac ischemic reperfusion injury. Mice were sacrificed 4 hours post-surgery, fixed by perfusion with 4% paraformaldehyde followed by PBS rinsing, then organs were explanted. Organs were then embedded in O.C.T, sectioned on a cryostat, and stained for nuclei and actin with FITC-Phalloidin. Imaging was performed on a Leica SP8 laser scanning confocal microscope and quantified using mEV uptake normalized to cell nuclei on ImageJ. Results: mEV uptake was significantly increased in scratch wounded cultures of both hDFs and MDCK cells (p<0.001), over uninjured control cells. Similarly, injured heart and skin tissues exhibited significantly increased exosomal uptake (p<0.001), relative to sham injury controls and skin tissues remote from the injury. Conclusions: Our experiments indicate that injured cells and tissues show increased uptake of milk derived exosomes, with in vitro data suggesting that this enhanced uptake occurs, at least in part, in a cell autonomous manner. The data also supports that our isolation protocol provides a novel mEV-based drug delivery system that may preferentially target injured tissues, including ischemic cardiac tissue.