A Monocyte Inspired Guiding System: Targeting of Exosomes to Ischemia Activated Microvascular Endothelium

Objectives: Exosomes derived from mesenchymal stem cells (MSCs) contain immunomodulatory and angiogenic factors that decrease remodeling after myocardial infarction. In clinical settings, repetitive delivery of exosomes is necessary due to the short half-life of exosomes. Multiple intravenous injections of exosomes in the early stages after MI are easy and minimally invasive. However, past studies have shown that intravenously injected exosomes accumulate in lung and liver.

We hypothesize that overexpression of monocyte adhesion molecules on MSC derived exosomes enables targeted delivery of exosome content to ischemia activated microvascular endothelium.

Methods: In this study, we aim to transiently overexpress E- and P-selectin specific ligands PSGL-1 and SLeX with modified mRNA (mmRNA) in adipose tissue derived MSCs (AT-MSCs). Additionally, luciferase is transiently expressed with mmRNA for in vivo tracking of exosomes. We hypothesize that exosomes isolated from these MSCs will express PSGL-1 and SLeX and will target ischemia induced microvascular endothelium in vitro and in vivo.

Results: As the first step for this project we aimed to create MSC derived exosomes that contain an in vivo tracking protein like luciferase. We successfully isolated exosomes from human AT-MSCs using a commercially available precipitation assay. Western blot analysis showed expression of CD63 and flotillin-1. Dynamic light scatter analysis demonstrated that isolated exosomes were within the expected size range of 131 ± 1.5 nm. After transient expression of luciferase we were able to demonstrate that exosomes exhibited 1.09 × 10⁷ ± 3.4 × 10⁵ RLU/µg protein of luciferase activity which compared to a luciferase activity of the primary cell lysate.

Conclusions: Targeted exosomes can be a powerful therapeutic platform to deliver various immunomodulatory and angiogenic miRNAs to limit post-ischemic remodeling. We have demonstrated that transient expression of proteins such as luciferase in MSCs results in expression of the same proteins in the exosome fraction of transfected cells.