Molecular interactions between bacterial lipids and Gemini surfactants: Insights from co-assembly of lipopolysaccharides with octenidine.

With a Gemini surfactant architecture and cationic 4-aminopyridinium headgroups, octenidine dihydrochloride (OCT) is one of the most potent antimicrobial actives. However, how its unique molecular architecture orchestrates interactions with lipopolysaccharides (LPS), a major constituent of the Gram-negative bacteria outer membrane, is not well understood. To probe OCT-LPS molecular interactions, we have studied the morphology and structure of nanoaggregates co-assembled between OCT and LPS. Furthermore, OCT was compared with Alkyl 8-10-8, an architecturally analogous Gemini surfactant with quaternary ammonium headgroups. Small-angle neutron scattering (SANS) revealed that, upon addition of 1.2 CMC OCT in the presence of 2.5 mM Ca, worm-like LPS-smooth micelles (of a radius ∼11.8 nm and a length ∼300 nm) transformed into longer rods with an ellipsoidal cross-section (9.4 nm × 29.3 nm) up to 3 µm in length. In contrast, co-assembly with 1.2 CMC Alkyl 8-10-8 led to a nano-aggregate mixture of lamellae and globules. Complementary cryo-TEM, dynamic light scattering, and zeta potential measurements further informed on the LPS-Gemini co-assembled nanostructures. To reconcile these observations, we suggest that the 4-aminopyridinium headgroups of OCT bind to anionic Lipid A phosphates in LPS through electrostatic, H-bonding, and π-system interactions, displacing bridging Ca ions. In contrast, the dimethylammonium headgroups of Alkyl 8-10-8 would undergo dynamic exchange with the Ca bridging between Lipid A phosphates, instead of binding to Ca, evident from disintegration of the worm-like aggregates. These findings provide mechanistic insights into the molecular interactions mediated by the OCT architecture, underpinning binding and subsequent disruption of the LPS-rich outer membrane leaflet in Gram-negative bacteria, with implications to future rational design of antimicrobial agents.