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

Year: 2026

Authors: Pacciani V., Cardellini J., Balestri A., Rojas-Rodriguez M., Calamai M., Tiboni M., Casettari L., Saunders C.E., Karimi A.A., Sanita G., Esposito E., Zendrini A., Radeghieri A., Paolini L., Bergese P., Montis C., Caselli L., Berti D.

Autors Affiliation: Univ Florence, Dept Chem Ugo Schiff, I-50019 Florence, Italy; Ctr Colloid & Surface Sci, CSGI, I-50019 Florence, Italy; European Lab Nonlinear Spect, I-50019 Sesto Fiorentino, Italy; Natl Res Council CNR INO, Natl Inst Opt, I-50125 Sesto Fiorentino, Italy; Univ Urbino Carlo Bo, Dept Biomol Sci, I-61029 Urbino, Italy; SPARTA Biodiscovery, London W12 7RZ, England; CNR, Inst Appl Sci & Intelligent Syst, EYE Lab, I-80131 Naples, Italy; Univ Brescia, Dept Mol & Translat Med, I-25123 Brescia, Italy; Univ Brescia, Dept Med & Surg Specialties, Radiol Sci & Publ Hlth, I-25123 Brescia, Italy.

Abstract: 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 in situ 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.

Journal/Review: ACS APPLIED MATERIALS & INTERFACES

Volume: 18 (4)      Pages from: 6530  to: 6548

More Information: This work has been supported by the European Community through the BOW Project (H2020-EIC-FETPROACT2019, ID 952183) and by PRIN 2022 PNRR: Lipid Nanovectors for the Delivery of Nucleic Acids: A Composition-Structure-Function Relationship Approach (Lancelot)-P2022RBF5P-CUP B53D23025810001-Finanziato dall’Unione Europea-Next Generation EU-Missione 4, Componente 2, Investimento 1.1-Avviso MUR D.D. 1409 del 14/09/2022. The authors also acknowledge MUR-Italy (Progetto Dipartimenti di Eccellenza 2018-2022, ref B96C1700020008 and Dipartimenti di Eccellenza 2023-2027 (DICUS 2.0) allocated to the Department of Chemistry Ugo Schiff). The University of Brescia also supported this work through Fondi ex 60%. We also thank the Center for Colloid and Surface Science (CSGI) for economic support. The authors thank the Elettra Sincrotrone Trieste (Trieste, Italy) for SAXS beamtime provision (Proposal No. 20247105, Austrian SAXS beamline), the imaging facility Unitech NOLIMITS (Universita degli Studi di Milano, Milan, Italy) and the Cryo-Electron Microscopy Laboratory of ISASI-CNR (Naples, Italy) for Cryo-EM imaging, as well as SPARTA Biodiscovery (U.K.) for single-particle Raman characterization. The authors also gratefully thank Camillo Almici and Arabella Neva of Bone Marrow Transplant (BMT) Unit of the A.O. Spedali Civili di Brescia for providing RBC concentrates.
KeyWords: microfluidic engineering; extracellular vesicles; hybrid lipid nanoparticles; cubosomes; redblood cell EVs
DOI: 10.1021/acsami.5c22972