Influence of the lipid thioether modification and formulation method on lipoplex structure, stability and...

Lipoplexes, formed by electrostatic interactions between nucleic acids and cationic lipids, must protect their cargo, enable cellular uptake and promote intracellular release for non-viral gene delivery. Although traditionally produced by bulk complexation, scalable microfluidic methods are now available. However, how lipid chemistry and formulation jointly influence physicochemical properties and biological performance remains poorly understood. To address this gap, we compared two structurally matched cationic lipids differing only in their hydrophobic chains: BSV107-S containing two thioether-functionalized chains, and BSV107, containing two mono-unsaturated chains. Each lipid was formulated either by complexing DNA with preformed liposomes obtained by thin-film hydration or by one-step DNA/lipid complexation through microfluidic assembly. We hypothesized that thioether incorporation would increase membrane fluidity and enhance intracellular plasmid release. Both lipids formed nanosized lipoplexes, with hydrodynamic diameters ranging 70-100 nm for microfluidic formulations and 100-140 nm for bulk-prepared lipoplexes at R ≥ 2. Cryo-TEM and SAXS revealed a multilamellar organization, with BSV107-S microfluidic lipoplexes showing larger interlamellar spacings than BSV107 or bulk-prepared systems. In HEK293T cells, transfection efficiency depended on the interplay between the lipid composition and formulation, with BSV107-S consistently outperforming BSV107 regardless of the formulation method. This improved performance correlated with increased membrane fluidity, measured by FRAP, supporting the role of thioether groups in modulating membrane dynamics. Together, these results establish structure-formulation-property relationships linking lipid chemical structure, formulation, physicochemical properties and biological performance, providing insights for the rational design of more efficient non-viral gene delivery systems.