On the mechanisms of internalization and intracellular delivery mediated by pH-sensitive liposomes

We investigated the molecular mechanisms by which pH-sensitive liposomes su rpass the cytoplasmic and endosomal membranes to deliver their aqueous cont ents into the cytoplasm. Various liposome formulations were evaluated for t heir efficacy to mediate intracellular delivery of encapsulated material, i ncluding a novel sterically stabilized pH-sensitive formulation ((DOPE: CHE M S: DSPE-PEG(2000) (6:4:0.3)) that was previously developed in our laborat ories. In an attempt to fully characterize the nature of liposome-cell inte ractions different approaches based on a dual-labeling fluorescence assay w ere used. Our results indicate that the efficacy of interaction of pH-sensi tive liposomes, both plain and sterically stabilized, with cells is strongl y determined by the inclusion of DOPE in their composition, independently o f the type of the amphiphilic stabilizer used. In fact, DOPE-containing lip osomes shown to be non-pH sensitive by biophysical assays, mediated cytopla smic delivery of their contents as efficiently as well known pH-sensitive f ormulations (e.g. DOPE:CHEMS). However, among the different formulations st udied, DOPE:CHEMS liposomes were those exhibiting the highest extent of cel l association. Moreover, our results with cells pretreated with metabolic i nhibitors or lysosomotropic agents clearly indicate that DOPE-containing li posomes are internalized essentially by endocytosis and that acidification of the endosomes is not the only mechanism involved in the destabilization of the liposomes inside the cell. (C) 2001 Elsevier Science B.V. All rights reserved.