Lipid nanoparticle shape and morphology determine the magnitude and duration of mRNA transfection
Lipid nanoparticles (LNP) are widely used for nucleic acid delivery, with current optimization efforts mainly focusing on refining formulation features, enabling extrahepatic targeting, and improving therapeutic efficacy. Particle shape and internal architecture are variables often overlooked in LNP design, although they critically influence particle stability, biodistribution, cellular uptake, and transfection. Here, we investigate how formulation and process parameters impact LNP shape, morphology, and internal structure, and how these structural features, in turn, influence mRNA loading, cell uptake, and expression. We demonstrate that modulation of lipid composition by increasing DSPC content drives a transition from compact spherical to vesicular non-spherical morphologies, with altered RNA loading at the single-particle level. While non-spherical LNP exhibited higher cellular uptake than spherical LNP, it was identified that this increase was relatively modest compared with the substantially higher and sustained protein expression for non-spherical structures. This suggests that increased uptake alone cannot explain the enhanced transfection performance. The trend was conserved across LNP formulations comprising clinically relevant ionizable lipids SM-102, ALC-0315 and DLin-MC3-DMA. Taken together, these findings establish LNP shape and internal morphology as functionally relevant design variables that should be considered in the design of next-generation LNP with improved mRNA delivery and expression.