Production of large unilamellar vesicles by a rapid extrusion procedure: characterization of size distribution, trapped volume and ability to maintain a membrane potential

Abstract

A technique for the rapid production of large unilamellar vesicles by repeated extrusion under moderate pressures (≤ 500 lb/in²) of multilamellar vesicles through polycarbonate filters (100 nm pore size) is demonstrated. In combination with freeze-thaw protocols where required, this procedure results in unilamellar vesicles with diameters in the range 60-100 nm and with trapped volumes in the region of 1-3 μl/μmol phospholipid. Advantages of this technique include the absence of organic solvents or detergents, the high lipid concentrations (up to 300 μmol/ml) that can be employed and the high trapping efficiencies (up to 30%) that can be achieved. Further, the procedure for generating these 'LUVET's' (large unilamellar vesicles by extrusion techniques) is rapid (≤ min preparation time) and can be employed to generate large unilamellar vesicles from a wide variety of lipid species and mixtures. As a particular illustration of the utility of this vesicle preparation, LUVET systems exhibiting a membrane potential (ΔΨ) in response to a transmembrane Na⁺/K⁺ gradient (K⁺ inside) have been characterized. By employing the lipophilic cation methyltriphenylphosphonium (MTPP⁺) it is shown that a K⁺ of diffusion potential (ΔΨ < -100 mV) forms rapidly in the presence of the K⁺ ionophore valinomycin for soya phosphatidylcholine (soya PC) LUVET's. The values of Δψ obtained correlate well with the K⁺ concentration gradient across the membrane, and it is demonstrated that the decay of Δψ with time depends on the flux of Na+ into the vesicles.