Investigation of lipid nanoparticle stability and integrity under isoelectric focusing stress conditions using taylor...

Liposomes and lipid nanoparticles (LNPs) are widely used as biomimetic systems and drug delivery vehicles, yet their isoelectric point (pI) remains underutilized as a characterization parameter. Capillary isoelectric focusing (cIEF) enables direct pI determination, but carrier ampholytes used in cIEF separations may compromise nanoparticle stability. Their influence on nanoparticle integrity remains poorly understood, hindering cIEF analysis development. Taylor dispersion analysis (TDA) is a particle-sizing technique that determines the hydrodynamic radius (R_h) of analytes, including nanoparticles and can be used for sensitive monitoring of nanoparticle stability.

TDA was used to characterize synthetic liposomes, lipid extract vesicles, and mRNA vaccine-derived LNPs in the presence of two carrier ampholyte (CA) systems, Pharmalyte 3-10 and AESlyte SH 3-10. Liver-derived vesicles exhibited pronounced ampholyte sensitivity, with rapid population loss and aggregation at relatively low CA concentrations, consistent with their failure in subsequent cIEF analysis. In contrast, POPC:DOPE:Chol liposomes and vaccine LNPs maintained nanoparticle populations across a broad CA concentration range, although concentration-dependent shifts in size distributions were observed. Both ampholyte systems enabled successful cIEF analysis of POPC:DOPE:Chol liposomes and vaccine LNPs. However, differences in nanoparticle stability were observed with different CA types at elevated concentrations.

This work demonstrates that TDA can serve as a predictive screening tool for evaluating ampholyte-induced lipid nanoparticle destabilization. By linking ampholyte concentration and type, vesicle composition, and electrophoretic focusing performance, the study identifies stability windows that support reliable pI determination of lipid-based nanoparticles.