Efficient cytoplasmic delivery of a fluorescent dye by pH-sensitive immunoliposomes

Abstract

We previously showed that liposomes composed of dioleoylphosphatidylethanolamine and palmitoyl-homocysteine (8:2) are highly fusion competent when ‘exposed to an acidic environment of pH <6.5. (Connor, J., M. B. Yatvin, and L. Huang, 1984, Proc. Natl. Acad. Sci. USA. 81:1715-1718). Palmitoyl anti-H2Kk was incorporated into these pHsensitive liposomes by a modified reserve-phase evaporation method. Mouse L929 cells (k haplotype) treated with immunoliposomes composed of dioleoylphosphatidylethanolamine/ palmitoyl-homocysteine (8:2) with an entrapped fluorescent dye, calcein, showed diffused fluorescence throughout the cytoplasm. Measurements by use of a microscope-associated photometer gave an approximate value of 50 µM for the cytoplasmic calcein concentration. This concentration represents an efficient delivery of the aqueous content of the immunoliposome. Cells treated with immunoliposomes composed of dioleoylphosphatidylcholine (pHinsensitive liposomes) showed only punctate fluorescence. The cytoplasmic delivery of calcein by the pH-sensitive immunoliposomes could be inhibited by chloroquine or by incubation at 20°C. These results suggest that the efficient cytoplasmic delivery involves the endocytic pathway, particularly the acidic organelles such as the endosomes and/or lysosomes. One possibility is that the immunoliposomes fuse with the endosome membranes from within the endosomes, thus releasing the contents into the cytoplasm. This nontoxic method should be widely applicable to the intracellular delivery of biomolecules into living cells. © 1985, Rockefeller University Press., All rights reserved.