3D-Printed Scaffolds Encapsulating Red Blood Cell Extracellular Vesicles for MicroRNA Delivery

Small non-coding RNAs (sncRNA) hold promising therapeutic potential. However, their clinical application is hindered by the poor cytocompatibility and limited transfection efficiency of conventional delivery vectors. In contrast, red blood cell-derived extracellular vesicles (RBCEVs) offer a safer, more efficient, and cost-effective alternative. Given the limited studies on the application of RBCEVs in the central nervous system (CNS) which is characterized by the presence of sensitive cell types with inherently low transfection efficiency, we hypothesized that RBCEVs could serve as a safe and effective sncRNA delivery vector for CNS applications, and that their incorporation into 3D-printed scaffolds could enable sustained and localized delivery of therapeutic sncRNAs. To test this, the uptake and gene silencing performance of RBCEVs were examined in primary CNS cell types, including astrocytes, neurons, oligodendrocyte precursor cells (OPCs), and microglia. While over 70% of OPCs and microglia internalized RBCEVs, uptake in neurons and astrocytes remained below 40%, indicating cell-type-specific uptake efficiency. Additionally, RBCEVs-mediated delivery of siRNA resulted in the highest gene knockdown efficiency in OPCs (74.2%), while triggering less than 30% gene knockdown in other cell types. Next, RBCEVs-encapsulated scaffolds were fabricated using digital light processing (DLP) 3D printing, enabling the sustained release of miR-219/miR-338-loaded RBCEVs for at least 21 days in vitro , which resulted in effective gene silencing that promoted OPC differentiation and myelination. Using spinal cord injury (SCI) as a proof-of-concept, scaffold-mediated delivery of RBCEVs-miR-219/miR-338 significantly promoted OPC differentiation and maturation in vivo as evidenced by increased CC1⁺ mature oligodendrocytes and reduced PDGFRα⁺ undifferentiated OPCs ( p < 0.001). Taken together, these results demonstrate the therapeutic potential of combining RBCEVs with DLP-printed scaffolds for localized and sustained sncRNA delivery in CNS disease treatment.