Engineering Hybrid Extracellular Vesicles for Functional mRNA Delivery

Abstract Despite the potential of extracellular vesicles (EVs) as nucleic acid delivery vectors, achieving efficient messenger RNA (mRNA) loading and cytoplasmic release of EV‐loaded mRNA cargo remains a critical challenge. Here, the properties of lipid nanoparticles (LNP) are explored to enhance mRNA loading and endosomal escape in human‐induced pluripotent stem cell EVs (hiPSC‐EVs), by generating EV‐LNP hybrids. Two EV‐LNP hybridization strategies are systematically evaluated: 1) cell‐based lipid nanoparticles (CELLNPs), formulated via microfluidic mixing of cell‐derived nanovesicles with a lipid mix, and 2) hybrid EVs (HEVs), formed through pH‐controlled fusion of EVs and LNPs. Both strategies are compared with passive endogenous mRNA loading (EndoEVs). Hybrid formation efficiency is assessed at the single‐particle level through nanoflow cytometry and Raman spectroscopy (SPARTA). In vitro efficacy is tested in a mCherry‐Galectin9 reporter cell line and embryonic stem cell‐derived cardiomyocytes. Single‐particle characterization reveals that a negligible fraction of EndoEVs contain mRNA cargo (<0.05%). CELLNPs achieve an average of 2.5% hybrid formation efficiency, whereas up to 73.3% HEVs are formed with optimized LNPs. Spectral signatures of HEVs indicate enrichment in cholesterol, proteins, and nucleic acids. HEVs effectively deliver mRNA in vitro, achieving protein expression at low particle doses. Overall, HEVs are an effective strategy to functionalize EVs for mRNA delivery.