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

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.