Membrane Modification of Extracellular Vesicles for Cardiac Disease Therapy

Myocardial infarction (MI) is the leading cause of mortality in developed countries, resulting in a major psychological and financial burden for society. Current treatments target rapid restoration of perfusion to limit damage to the heart, rather than promoting tissue regeneration and subsequent contractile function recovery. Regenerative therapies, including stem cells, tissue grafts and extracellular vesicles (EVs), surge as a promising alternative, however, their efficacy is severely limited by low long-term cell survival, and poor homing and engraftment to the myocardium. Different strategies to overcome these problems include genetic-modification or membrane surface modification of the transplanted cells, and by their incorporation onto soft biomimetic materials.¹<br/><br/>Inspired by the Artificial Membrane Binding Proteins (AMBPs) technology previously developed within our group,² we have designed a novel versatile system comprised of two bio-orthogonal reacting pairs. First, a surfactant-coated protein fusion that anchors to bilayer lipid membranes and, second, a protein fusion that reacts selectively with the former³ and contains another protein or peptide that enhances the properties of artificial extracellular vesicles (AEVs), which can be used as carriers of therapeutic payloads. After confirming the biophysical interaction between the reacting AMBPs in solution, we have demonstrated that the interaction between the reacting AMBPs also occurs when embedded on the membranes of in-house built AEVs <i>via</i> liposome extrusion. Stability studies with dynamic light scattering (DLS) revealed that vesicles in our AMBP-AEV system were intact for up to a week.<br/><br/>We then selected a cardiac-homing protein to be fused to the second counterpart of the system, obtaining approximately a 20-fold uptake increase for fibronectin-rich cell types statically, upon no detriment to cell viability. Further experiments under physiologically relevant shear stress (2 dynes) resulted in a 10-fold increase of the desired AMBP-AEV uptake and retention. Additionally, we demonstrated exclusive localization of the cardiac-homing AMBP-AEV system in zebrafish hearts, highlighting their potential as therapeutic in cardiac disease. The extrusion-based AEV synthesis facilitates their loading with a cargo, for which we employed mCherry, a red-fluorescent protein, as a payload inside the vesicles to demonstrate only AMBP-modified red AEVs were uptaken <i>in vitro</i> and <i>in vivo</i>.<br/><br/>In conclusion, we have shown cellular uptake of AMBP-AEVs and the possibility of directing these to a specific target such as the heart. We demonstrated our cardiac homing AMBP-AEV system to function under physiological conditions (2 dyne shear stress), which localised exclusively in zebrafish hearts. This constitutes a plug-and-play system that can be readily modified or repurposed depending on the desired outcome for regenerative therapies and can be incorporated in any bilayer membrane.<br/> <br/> <br/>(1) Cruz-Samperio, R.; Jordan, M.; Perriman, A. Cell Augmentation Strategies for Cardiac Stem Cell Therapies. <i>Stem Cells Transl. Med.</i> <b>2021</b>, <i>10</i> (6), 855–866. https://doi.org/10.1002/sctm.20-0489.<br/>(2) Xiao, W.; Green, T. I. P.; Liang, X.; Delint, R. C.; Perry, G.; Roberts, M. S.; Le Vay, K.; Back, C. R.; Ascione, R.; Wang, H.; Race, P. R.; Perriman, A. W. Designer Artificial Membrane Binding Proteins to Direct Stem Cells to the Myocardium. <i>Chem. Sci.</i> <b>2019</b>, <i>10</i> (32), 7610–7618. https://doi.org/10.1039/c9sc02650a.<br/>(3) Hatlem, D.; Trunk, T.; Linke, D.; Leo, J. C. Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins. <i>Int. J. Mol. Sci.</i> <b>2019</b>, <i>20</i> (9). https://doi.org/10.3390/ijms20092129.