Multiphasic lipid nanoparticles: structural heterogeneity drives endosomal bypass for enhanced RNA delivery

Year: 2026

Authors: Conti L., Rojas-Rodriguez M., Caselli L., Bonavolonta D., Sanita G., Esposito E., Calamai M., Marradi M., Berti D., Montis C.

Autors Affiliation: Univ Florence, Dept Chem Ugo Schiff, I-50019 Sesto Fiorentino, Florence, Italy; Ctr Colloid & Surface Sci, CSGI, I-50019 Sesto Fiorentino, Florence, Italy; European Lab Nonlinear Spect, Via Nello Carrara 1, I-50019 Sesto Fiorentino, Italy; CNR, Inst Appl Sci & Intelligent Syst, EYE Lab, Via Pietro Castellino 111, I-80131 Naples, Italy; Natl Res Council CNR INO, Natl Inst Opt, I-50125 Sesto Fiorentino, Italy.

Abstract: Lipid nanoparticles (LNPs) are leading vectors for nucleic acids (NA) delivery. However, inefficient endosomal escape remains a critical bottleneck, with typically less than 3% of internalized NA reaching the cytosol. While clinically approved LNPs rely on pH-triggered protonation of ionizable lipids to destabilize endosomal membranes, growing evidence suggests that the internal nanostructure of LNPs may also play an active role in mediating intracellular delivery. Here, we introduce a new design strategy that harnesses topological mismatch to engineer structurally active LNPs with a highly heterogeneous, multiphasic core architecture. Using glycerol monooleate (GMO)-a curvature-promoting monoglyceride-as the base lipid, we incorporated cationic and ionizable functional lipids to drive phase separation into topologically distinct domains, arising by a combination of geometric and electrostatic packing asymmetry. Cryo-electron microscopy and synchrotron small-angle X-ray scattering revealed composition-dependent multiphasic organization, offering mechanistic insight into phase-separating behavior. We further demonstrated that increased internal heterogeneity correlates with enhanced NA cytosolic release and transfection efficiency. Unlike conventional LNPs, multiphasic LNPs deliver the cargo via direct plasma membrane fusion, enabling efficient cytosolic release while bypassing endosomal entrapment. By leveraging phase separation, this work introduces new, structurally active LNPs and provides a blueprint for engineering NA delivery vectors with improved intracellular delivery.

Journal/Review: JOURNAL OF COLLOID AND INTERFACE SCIENCE

Volume: 709      Pages from: 139966-1  to: 139966-17

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 (Lance-lot)-P2022RBF5P-CUP B53D23025810001-Finanziato dall’Unione europea-Next Generation EU-Missione 4, Componente 2, I nvestimento 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). M.M., C.M., L.C. also acknowledge co-funding from the European Union-NextGenerationEU programme in the context of the National Recovery and Resilience Plan, Mission 4, Component 2, Investment 1.4, CN00000041, CN3 National Center for Gene Therapy and Drugs based on RNA Technology-Spoke 5 Inflammatory and Infectious Diseases, CUP: B13C22001010001. This work also received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 956977. M.M. acknowledges support from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement 101008072 (H2020-MSCA-RISE-2020 Project SUPRO-GEN Supramolecular Polyamine Gene Vectors for Cancer Therapy) . E.E. and G.N. also thank the project PON IMPARA PIR01_0023 (support for Cryo-electron microscopy, EYE LAB laboratory, ISASI-CNR, Naples) . We also thank the Center for Colloid and Surface Science (CSGI) for economic support. The authors thank theElettra Sincrotrone Trieste (Trieste, Italy (Proposal No. 20242174, Austrian SAXS beamline) ) and the European Synchrotron Radiation Facility (ESRF) (Proposal No. SC-5534, ID02 beamline) for SAXS beamtime provision, 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.
KeyWords: Phase-separation; Lipid nanoparticles; Multiphase nanoparticles; Lipid mesophases; Gene delivery; Non-lamellar lipid nanoparticles
DOI: 10.1016/j.jcis.2026.139966