Biophysical studies and intracellular destabilization of pH‐sensitive liposomes

We examined changes in membrane properties upon acidification of dioleoylphosphatidylethanolamine/cholesterylhemisuccinate liposomes and evaluated their potential to deliver entrapped tracers in cultured macrophages. Membrane permeability was determined by the release of entrapped calcein or hydroxypyrene‐1,3,6‐trisulfonic acid (HPTS)‐ p ‐xylene‐bis‐pyridinium bromide (DPX); membrane fusion, by measuring the change in size of the liposomes and the dequenching of octadecylrhodamine‐B fluorescence; and change in lipid organization, by 31 P nuclear magnetic resonance spectroscopy. Measurement of cell‐associated fluorescence and confocal microscopy examination were made on cells incubated with liposomes loaded with HPTS or HPTS‐DPX. The biophysical studies showed (i) a lipid reorganization from bilayer to hexagonal phase progressing from pH 8.0 to 5.0, (ii) a membrane permeabilization for pH<6.5, (iii) an increase in the mean diameter of liposomes for pH<6.0, and (iv) a mixing of liposome membranes for pH<5.7. The cellular studies showed (i) an uptake of the liposomes that were brought from pH 7.5–7.0 to 6.5–6.0 and (ii) a release of ∼15% of the endocytosed marker associated with its partial release from the vesicles (diffuse localization). We conclude that the permeabilization and fusion of pH‐sensitive liposomes occur as a consequence of a progressive lipid reorganization upon acidification. These changes may develop intracellular after phagocytosis and allow for the release of the liposome content in endosomes associated with a redistribution in the cytosol.