Biotinylated Red Blood Cell-derived Extracellular Vesicles as a promising tool for targeted delivery

Red blood cell-derived extracellular vesicles (RBCEVs) are promising biologics delivery carriers due to their high biocompatibility, absence of DNA, and easy availability from donors or patients. RBCEVs can efficiently deliver their cargo to recipient cells, exerting specific biological effects. Moreover, their membrane can be engineered for targeted delivery to particular cells or tissues with biotinylation, a safe and biocompatible method already applied in clinics. This study explores RBCEV’s biotinylation to enhance targeting specificity toward cardiomyocytes. NHS-biotin was used to biotinylate not dialysed (ND) and engineered RBCs. Unloaded (UL) and loaded (L) RBCs underwent hypotonic dialysis with or without the addition of cargo (i.e., FITC-dextran or miRNA mimics), respectively. Biotinylation efficiency was assessed by FC using Alexa Fluor-streptavidin. ND, UL, and L RBCs were then induced to vesiculate via the patented soft extrusion method. The resulting biotinylated RBCEVs (B-RBCEVs) were characterised by DLS, NTA and FC, following MISEV2023 guidelines. Additional FC analyses quantified biotin incorporation on EVs membrane. In vitro studies will assess differential RBCEV and B-RBCEV uptake in cardiomyocytes, exploiting an avidin bridge with a Cx43 biotinylated Ab. To do this, several cell cultures (e.g., AC16, H9C2, HEK293, HeLa) will be tested. Finally, cargo delivery and efficacy will be tested in several cell types. This study confirms successful membrane biotinylation in both ND and engineered RBCs. For the first time, high biotinylation efficiency and satisfactory recovery rates were achieved in UL and L RBCs. FC showed comparable biotin levels across all groups, suggesting that the loading procedure preserves membrane integrity. Soft extrusion yielded RBCEVs with good physical and biological features, as shown by FC and NTA analyses. Also in this case, FC showed high biotin retention on RBCEV membranes, mirroring their respective mother cells. The in vitro uptake of B-RBCEVs by cardiomyocytes via the biotin-Cx43 Ab-avidin bridge, and their efficacy in cargo release are currently under investigation. This work presents a strategy for RBCEV surface functionalization while preserving native properties, for targeted delivery to specific cells or tissues. These data pave the way for future in vitro and in vivo studies to evaluate the B-EV potential in targeted therapies, particularly cardiovascular diseases.