Uphill Accumulation of Ionic Species into a Lipid Vesicle by the Concentration Gradient of Counter Ions

The "uphill (against the concentration gradient)" accumulation of a hydrophobic cation (rhodamine 6G, R6G⁺) into the inner phase of a giant unilamellar vesicle (GUV) was realized with the concentration gradient of the counter anion (X^- = ClO₄^-, BF₄^-, or Br^-) in the presence of phosphate buffer (P^-, pH = 7) in the inner and outer phase of the GUV and detected as the increase of the R6G⁺ fluorescence intensity in the inner phase using a confocal laser scanning fluorescence microscope. The addition of X^- in the outer phase of the GUV caused the accumulation of R6G⁺ in the inner phase. The degree and kinetics of the accumulation were dependent on the concentration and type of X^-; e.g., the inner concentration of R6G⁺ reached 2.5 times that in the outer phase of GUV after adding 10 mM ClO₄^-. The accumulation was theoretically simulated by assuming the distribution of ion pairs (R6G⁺ and X^-, R6G⁺, and P^-) between the aqueous phase and the lipid bilayer membrane (ion pair distribution model) and the transmembrane fluxes of R6G⁺, X^- and P^-. The theoretical simulation rationalized the accumulation degree and kinetics of the experimental results. The accumulation of the target cation by the concentration gradient of the counter anion demonstrated in this study can be an effective method for the preparation of liposomal drugs.