pH-sensitive liposomes : characterization and application

It has been demonstrated that liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and palmitoylhomocysteine (PHC) have the ability to fuse with adjacent membranes upon exposure to mildly acid pH. This phenomenon was shown using resonance energy transfer, gel filtration, and electron microscopy. The ability of liposomes to fuse is absolutely dependent on the presence of DOPE and a weakly acidic amphiphile. The acid induced fusion event is a leaky process, but the leakage can be reduced by 50%, with only a small loss of fusion ability, by the inclusion of 40 mole percent cholesterol. Using an established monoclonal antibody targeting system (Huang et al. 1983 JBC 258, 14034), pH-sensitive immunoliposomes were prepared which successfully delivered entrapped calcein to the cytoplasm of target cells. The addition of chloroquine, which raises the internal pH of cellular vacuoles, blocks the cytoplasmic delivery by the pH-sensitive immunoliposomes. pH-insensitive immunoliposomes delivered calcein only to the endosome/lysosome system and not the cytoplasm. Liposomes composed of DOPEroleic acid (OA) have also been demonstrated to be fusogenic at mildly acidic pH. Using resonance energy transfer, a synergistic effect in the ability to promote fusion was established between protons and the divalent cations Ca⁺² and Mg ⁺². ³¹P-NMR and light scattering of DOPE:OA liposomes under acidic conditions demonstrate that the effect of the protons and the divalent cations is to force the DOPE to revert to the hexagonal II configuration. Using DOPE:OA, pH-sensitive immunoliposomes successfully delivered the cytotoxic drugs Ara-C and MTX to the cytoplasm of target cells. Furthermore the pH-sensitive immunoliposomes are more effective than free drug or pH-insensitive immunoliposomes. The lysosomotrophic amines, chloroquine and NH₄Cl both were able to block the pH-sensitive immunoliposome delivery by raising the internal pH of endosomes/lysosomes. In vivo experiments with DOPE:OA immunoliposomes indicate that the liposomes rapidly aggregate and release their contents upon exposure to plasma. These results indicate that pH-sensitive immunoliposomes are an effective tool for in vitro cytoplasmic delivery but are ineffective for in vivo delivery at this point in development.