Phenolic terpenes in liposomal bilayers: Unraveling physicochemical interactions and membrane perturbation via biophysical and computational approaches

The interaction of small hydrophobic and amphiphilic molecules with lipid membranes plays a pivotal role in modulating membrane structure, dynamics, and permeability, which are key factors in applications ranging from drug delivery to antimicrobial design. In this study, we investigate the incorporation of the phenolic monoterpenes thymol and carvacrol, along with some ether derivatives, into unilamellar liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). These systems were prepared via the thin-film hydration method and characterized at two distinct terpene/lipid molar ratios. A comprehensive array of biophysical techniques was employed to elucidate the structural and thermotropic modifications induced by terpene incorporation. Dynamic light scattering (DLS) provided insights into vesicle size and colloidal stability. Differential Scanning Calorimetry (DSC) revealed perturbations in membrane phase behaviour, while Raman spectroscopy offered molecular-level details on lipid-terpene interactions, particularly at the headgroup and upper acyl chain regions. Additionally, Molecular Dynamics simulations indicated a preferential localization of terpenes just beneath the lipid headgroup, further reinforcing the experimental observations. Fluorescence-based permeability assays using 5(6)-carboxyfluorescein (CF), interpreted through a biphasic kinetic model, showed that phenolic terpenes promoted membrane leakage, especially during the initial burst phase, suggesting a key role for the phenolic -OH group in mediating interfacial interactions. This study advances our understanding of molecular determinants governing guest-bilayer interactions and underscores the utility of model membrane systems and complementary biophysical tools for probing structural phenomena in soft matter systems.