Single-Polynucleotide Encapsulation in Lipid Nanoparticles via Single-Step Assembly at Physiological pH
- 1Laboratoire de Génie Chimique
- 2Université de Toulouse, CNRS, INPT, UPS
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.
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BibTeX
@misc{guichonnet2026singlepolynucleotide,
title = {{Single-Polynucleotide Encapsulation in Lipid Nanoparticles via Single-Step Assembly at Physiological pH}},
author = {Maxime Guichonnet and Johanna Mirande-Ney and Anne-Sophie Kirstetter and Martine Meireles and Sébastien Teychené and Kevin Roger},
howpublished = {ChemRxiv},
year = {2026},
month = oct,
doi = {10.26434/chemrxiv.15009963/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009963/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Single-Polynucleotide Encapsulation in Lipid Nanoparticles via Single-Step Assembly at Physiological pH
AU - Maxime Guichonnet
AU - Johanna Mirande-Ney
AU - Anne-Sophie Kirstetter
AU - Martine Meireles
AU - Sébastien Teychené
AU - Kevin Roger
PY - 2026
DA - 2026/10/06
PB - ChemRxiv
DO - 10.26434/chemrxiv.15009963/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009963/v1
AB - 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.
ER -