Cytosolic delivery of macromolecules 4. Head group-dependent membrane permeabilization by pH-sensitive detergents.

Three tertiary amine-based detergents with zero, one, or two hydroxyl groups at various positions in their head group were characterized for their ability to promote the cytosolic delivery of macromolecules. Critical micellar concentrations (CMC) and membrane-bound pKa values of the lipid constructs increased with increasing head group polarity, ranging from 1-5 microM and 5.9 to 6.3, respectively. Fluorescence resonance energy transfer (FRET) and calcein leakage experiments revealed that when the amine group is protonated introduction of -OH moieties to detergent head groups enhanced their ability to interact with and permeabilize anionic, endosome-mimicking vesicles. Different formulations of a diethanolamine-based lipid (DEL) were further evaluated for pH-dependent hemolytic activity and ability to promote cytosolic delivery of macromolecules in vitro. Intact liposomes containing DEL at its maximum limit of incorporation were less efficient than DEL-containing micelles in promoting hemoglobin leakage from human erythrocytes at acidic pH. In HeLa cells, DEL-containing detergent micelles facilitated efficient cytosolic release of endocytosed macromolecules such as fluorescein-labeled dextran of MW 10 kDa. This observation was further corroborated by a functional assay based on antisense-mediated up-regulation of enhanced green fluorescent protein (EGFP). Taken together, our findings emphasize the key role of polar head groups and micellar architecture of pH-sensitive detergents in mediating endosomal permeabilization and the efficient cytosolic delivery of macromolecules.