Kinetic analysis of endocytosis and intracellular fate of liposomes in single macrophages.

Endocytosis and the intracellular fate of liposomes in single mouse peritoneal macrophages were examined kinetically by fluorescence microphotometry. Liposomes labeled with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine or containing 8-amino-naphthalene-1,3,6-trisulfonate were promptly incorporated into macrophages on incubation at 37 degrees C, but fluorescence increase caused by hydrolysis of 4-methylumbelliferyl-beta-D-glucoside encapsulated in the liposomes was observed after 30 min of incubation. The fluorescences of calcein and 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS) in liposomes, which were respectively quenched statically due to high concentration and dynamically by a co-entrapped fluorescence quencher, p-xylene-bis-pyridinium bromide, also increased from 30 min after the start of liposome incorporation, indicating that macrophages require this period for intracellular delivery of liposomes from the cell surface to lysosomes. Measurement of the intraendosomal pH change in a single macrophage at 37 degrees C with liposomes containing a pH-sensitive fluorescent marker, HPTS, showed that the pH value decreased continuously to a constant value of 5.5 in 30-40 min after endocytosis, and this decrease was reversed on addition of NH4Cl, suggesting that acidification of endosomes is not a stepwise reaction and is coupled with delivery of liposomes. These fluorescence microphotometric systems using liposomes containing different fluorescent dyes should be useful for kinetic analyses of the endocytosis and intracellular fate of liposomes in various phagocytes.