Uphill Accumulation of Anionic Species in Lipid Vesicles Driven by the Concentration Gradient of Hydrophilic Cations

Ion permeation through bilayer lipid membranes (BLMs) is a fundamental process governing the transport of ionic drugs across biological membranes. In this study, we experimentally demonstrated that water-soluble but hydrophobic anions (HS-BODIPY^2-) can be accumulated inside giant unilamellar vesicles (GUVs) by utilizing concentration gradients of driving cations (X⁺). By varying the cation species (X⁺ = K⁺, Na⁺, CH₃NH₃⁺, (CH₃)₂NH₂⁺, (CH₃)₃NH⁺, and (CH₃)₄N⁺), the accumulation of HS-BODIPY^2- strongly depended on both the species and concentration of X⁺. Hydrophilic cations such as Na⁺ and K⁺ also acted as driving ions in neutral GUVs, likely through affinity with phosphate or carbonyl groups of phospholipid headgroups. Furthermore, incorporation of cationic lipids enhanced the accumulation of HS-BODIPY^2-, whereas anionic lipids had little effect. These findings demonstrate that ion-pair distribution into BLMs and cation-lipid interactions play crucial roles in governing membrane permeability of ionic species. The mechanism revealed here offers a novel and biocompatible strategy for controlling ion transport and encapsulating anionic drugs such as nucleic acids and nucleotides into liposomal systems.