Mechanistic insights from dynamic structural changes of model bacterial cytoplasmic membranes challenged by Gemini surfactant octenidine

Octenidine dihydrochloride (OCT) is a Gemini surfactant that is highly potent in bacterial membrane disruptions via a multitude of molecular interactions mediated by its cationic 4-aminopyridinium headgroup and hydrophobic tail and spacer groups. However, the mechanistic details for such interactions remain to be fully understood. Here, structural changes of unilamellar ternary liposomes comprising 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG) and cardiolipin (CL), mimicking the cytoplasmic membrane of E. coli bacteria, challenged by OCT at different lipid:surfactant molar ratios (50:1–1:1) and different temperatures (25, 40, and 70 °C), have been studied using synchrotron time-resolved stopped flow small-angle X-ray scattering (Tr-SAXS), complemented by wide-angle X-ray scattering (WAXS), dynamic light scattering and zeta potential measurements. The high temporal resolution of the scattering data offered insights into the breakdown-reassembly mechanism of liposome disruption, involving initial membrane lysing and subsequent lipid re-assembly into multilamellar stacks, which then collapsed into a condensed multilamellar L α C phase and a cubic Pn3m phase upon heating. Critical comparisons between OCT and Alkyl 8-10-8, a structurally analogous Gemini surfactant differing only with its quaternary ammonium headgroups, revealed further mechanistic insights from subtle differences in the lipid-surfactant structures. Compared to Alkyl 8-10-8, OCT mediated membrane disruption at a lower concentration, perturbing the membrane thickness and altering the membrane gel-to-fluid phase transition temperature, also causing membrane surface charge neutralisation and reversal. These findings shed light on molecular mechanism of OCT-membrane interactions and the role of the OCT headgroup architecture in its antimicrobial efficacy. • Octenidine dihydrochloride (OCT), a cationic Gemini surfactant, is a highly effective broad-spectrum antiseptic. • The underlying mechanism for OCT bacterial membrane disruption is not fully understood. • The high temporal resolution of TR-SAXS offers insights into the dynamic breakdown-reassembly mechanism of liposome disruption. • Comparison between OCT and Alkyl 8–10-8 (a Gemini analogue) highlights the impact of subtle difference in surfactant architectures. • Compared to Alkyl 8–10-8, OCT disrupts membranes at a lower concentration and alters the membrane gel-to-fluid phase transition temperature.