Single-Polynucleotide Encapsulation in Lipid Nanoparticles via Single-Step Assembly at Physiological pH
Year: 2026
Type: preprint
Abstract: Lipid nanoparticles (LNPs) are the leading nonviral platform for polynucleotide delivery, but structural heterogeneity and poorly controlled loading states limit quantitative structure–function analysis and rational formulation design. Standard protocols use rapid mixing under acidic conditions followed by buffer exchange, reflecting the assumption that ionizable lipid protonation is required for nucleic acid complexation. Here, we show that lipid–polynucleotide nanoparticles can instead be assembled directly at near-physiological pH through a single rapid micromixing step. Under near-stoichiometric lipid-to-polynucleotide conditions, rapid solvent exchange yields a homogeneous nanoparticle population without acidic formulation or post-assembly pH adjustment. Small-angle X-ray and neutron scattering (SAXS and SANS), together with cryogenic transmission electron microscopy (cryo-TEM), reveal a common core–multishell architecture consistent with encapsulation of one polynucleotide strand per particle. In situ SAXS shows that the final structure is established within submillisecond timescales during solvent exchange, indicating rapid cooperative coassembly rather than postmixing restructuring. Systematic variation of mixing conditions, stoichiometry, PEGylated lipid content, ionic strength, and pH demonstrates that this structural regime is selected by the assembly pathway rather than composition alone. The nanoparticles remain structurally stable over extended periods and retain intracellular delivery activity, while providing a constrained system for probing formulation-dependent effects. These results establish physiological-pH assembly as a route to pathway-selected single-polynucleotide nanoparticles and identify loading state as a design variable alongside composition, size, and surface chemistry. More broadly, they highlight assembly history as a determinant of nanoparticle structure and pathway-directed assembly as a strategy for controlling soft nanomaterial heterogeneity at its origin rather than after formation.
Source: ChemRxiv
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7220737832