Factors affecting solute entrapment in phospholipid vesicles prepared by the freeze-thaw extrusion method: a possible general method for improving the efficiency of entrapment.

It is often assumed that the internal solute concentrations of phospholipid vesicles are equal to those in the medium in which they were prepared, particularly when freeze-thaw cycles are employed during the procedure. Conditions are reported here which when used to prepare vesicles by the polycarbonate filter extrusion method, produce approximately 12- and approximately 7-fold higher internal concentrations of Ca2+ and sucrose, respectively, than exist in the external medium. Formation of these large gradients is dependent upon the use of freeze-thaw cycles during preparation, on the presence of tetraethylammonium perchlorate in the medium, and is independent of media pH across the region of pH 5-9. Gradient formation is antagonized by high concentrations of an impermeant solute (NaCl). It is proposed that gradients form because solutes are concentrated by exclusion from ice during freezing but that they are normally dissipated by osmotic lysis during thawing. The presence of a permeant solute such as tetraethylammonium perchlorate provides an alternative mechanism to balance osmotic pressure, thereby preserving the gradients of impermeable species.