Zn²⁺ Induced Irreversible Aggregation, Stacking, and Leakage of Choline Phosphate Liposomes

The interaction between lipids and metal ions is important for metal sensing, cellular signal transduction, and oxidative lipid damage. While most previous work overlooked the phosphate group of lipids for metal binding, we herein highlight its importance. Phosphocholine (PC) and its headgroup inversed choline phosphate (CP) lipids were used to prepare liposomes. From dynamic light scattering (DLS), Zn²⁺ causes significant aggregation or fusion of the CP liposomes, but not PC liposomes. The size change induced by Zn²⁺ is not fully reversed by adding EDTA, implying liposome fusion induced by Zn²⁺. Isothermal titration calorimetry (ITC) shows that binding between Zn²⁺ and CP liposomes is endothermic with a K_d of 110 μM Zn²⁺, suggesting an entropy driven reaction likely due to the release of bound water. In comparison, no heat was detected by titrating Zn²⁺ into PC liposomes or Ca²⁺ into CP liposomes. Furthermore, Zn²⁺ causes a transient leakage of the CP liposomes, and further leakage is observed upon removing Zn²⁺ by EDTA. Transmission electron microscopy (TEM) with negative stained samples showed multilamellar CP lipid structures attributable to Zn²⁺ sandwiched between lipid bilayers, leading to a proposed reaction mechanism. This work provides an interesting system for studying metal interacting with terminal phosphate groups in liposomes, affecting the size, charge, and membrane integrity of the liposomes.