Ethanol-Guided Hybridization of Extracellular Vesicles with Liquid-Crystalline Lipid Nanoparticles

Hybrid nanosystems that integrate biological and synthetic lipid assemblies hold great promise for tailoring nanoscale interfaces with programmable chemical and structural functionality. However, existing approaches to hybridize extracellular vesicles (EVs) with lipid nanoparticles (LNPs) compromise either the EV bioactivity or the native supramolecular organization of synthetic LNPs, undermining structure-dependent functionality. Here, we introduce an ethanol-mediated microfluidic assembly route that enables the <i>in situ</i> formation and hybridization of nonlamellar liquid-crystalline lipid nanoparticles (LCNPs) with red-blood-cell-derived EVs (RBCEVs) in a single step. This process exploits ethanol-induced interfacial reorganization to drive EV incorporation without compromising the LCNP cubic architecture. Synchrotron small-angle X-ray scattering (SAXS) and cryogenic electron microscopy reveal hybrid nanoparticles that retain long-range cubic order, with RBCEV membrane proteins localized within phase-segregated nanodomains. Single-particle Raman analysis and enzymatic assays confirm molecular-level hybridization and preserved EV biofunctionality. Hybrid LCNPs also exhibit enhanced uptake in HEK293t cells. Mechanistic SAXS studies uncover that ethanol transiently stabilizes a swollen sponge-like intermediate, which mediates controlled fusion and acts as a structural template upon solvent removal, imparting long-lasting structural stability. This study elucidates the physicochemical mechanism of ethanol-guided hybridization between biogenic systems and soft nanostructured colloids, establishing design principles for structurally controlled nanohybrids with broad applicability in nanomedicine.