Biotinylated RBCEVs as a promising tool for surface functionalization and targeting

Introduction: Red blood cells-derived extracellular vesicles (RBCEVs) have recently emerged as promising tools for targeted drug and biologics delivery. Cargo-loaded RBCEVs can be efficiently produced starting from preloaded RBCs using the innovative “soft extrusion” technique. To date, the produced RBCEVs have been employed in both in vitro and in vivo studies, leveraging their inherent uptake and biodistribution characteristics. Additionally, biotinylation is a fruitful strategy for attaching various peptides and antibodies to cell or EV surfaces, enabling specific targeting of particular cell types or tissues. This method is fully biocompatible and safe, so it has already found applications in clinical settings. The present work investigates the biotinylation of RBCEVs as a method for membrane engineering and targeted delivery, focusing on enhancing specificity for cardiomyocytes. Methods: Two biotin derivatives, NSH-biotin and NSH-LC-biotin, were employed to biotinylate RBCEVs. Both the efficiency of biotinylation and cell recovery were assessed. Furthermore, flow cytometry analyses with Alexa Fluor-streptavidin were conducted to quantify the degree of biotin incorporation. A three-step delivery system was employed to target cardiomyocytes. It consists in the administration of the biotin-Cx43 Ab followed by the introduction of avidin, which forms a stable complex with the Ab. Finally, biotinylated RBCEVs were decorated with the specific antibody biotin-Cx43 using an avidin bridge and administered to allow them to attach via avidin’s biotin-binding sites. Results: Both biotinylation strategies demonstrated high efficiency and satisfying cell recovery rates. Flow cytometry analysis confirmed successful biotin incorporation on RBCEVs, facilitating specific targeting of cardiomyocytes via the biotin Cx43 Ab-avidin bridge. The functionalization of RBCEVs with the antibody resulted in enhanced binding to target cells, highlighting the effectiveness of this approach for directed delivery. Conclusion: This study proposes the exploitation of a three-step methodology for the biotinylation and functionalization of RBCEVs, enabling targeted delivery to cardiomyocytes. The successful application of this technique has great potential to advance therapeutic strategies in cardiovascular diseases, paving the way for future in vitro and in vivo studies to assess the potential of biotinylated EVs in targeted therapies and to optimize this delivery system for clinical applications.