KINETIC-ANALYSIS OF ENDOCYTOSIS AND INTRACELLULAR FATE OF LIPOSOMES IN SINGLE MACROPHAGES

Endocytosis and the intracellular fate of liposomes in single mouse pe ritoneal macrophages were examined kinetically by fluorescence microph otometry. Liposomes labeled with -2,1,3-benzoxadiazol-4-yl)phosphatidy lethanolamine or containing 8-amino-naphthalene-1,3,6-trisulfonate wer e promptly incorporated into macrophages on incubation at 37 degrees C , but fluorescence increase caused by hydrolysis of 4-methylumbellifer yl-beta-D-glucoside encapsulated in the liposomes was observed after 3 0 min of incubation. The fluorescences of calcein and 8-hydroxy-1,3,6- pyrenetrisulfonate (HPTS) in liposomes, which were respectively quench ed statically due to high concentration and dynamically by a co-entrap ped fluorescence quencher, p-xylene-bis-pyridinium bromide, also incre ased from 30 min after the start of liposome incorporation, indicating that macrophages require this period for intracellular delivery of li posomes from the cell surface to lysosomes. Measurement of the intraen dosomal pH change in a single macrophage at 37 degrees C with liposome s containing a pH-sensitive fluorescent marker, HPTS, showed that the pH value decreased continuously to a constant value of 5.5 in 30-40 mi n after endocytosis, and this decrease was reversed on addition of NH4 Cl, suggesting that acidification of endosomes is not a stepwise react ion and is coupled with delivery of liposomes. These fluorescence micr ophotometric systems using liposomes containing different fluorescent dyes should be useful for kinetic analyses of the endocytosis and intr acellular fate of liposomes in various phagocytes.