Biochemistry zyxwvut 1990, 29, zyxwvu 4929-4939 4929 Endocytosis and Intracellular Fate of Liposomes Using Pyranine as a Probe? Robert M. Straubinger,*,f Demetrios Papahadjopoulos,*~~~lI and Keelung Hong**ll Department of Pharmacology and Cancer Research Institute, University of California, San Francisco, California 94143, and Department of Pharmaceutics, University at Buffalo, State University of New York, Amherst, New York 14260 Received October 16, 1989; Revised Manuscript Received January 8, 1990 ABSTRACT: Lipid vesicles (liposomes) containing pH-sensitive fluorophores were used as probes for the study of liposome entry and intracellular fate. Pyranine [8-hydroxy- zyxwv 1,3,6-pyrenetrisulfonate (HPTS)] was entrapped in the liposome aqueous core during preparation to provide a means of detecting internalization into living cells. HPTS is highly water soluble and shows a strong pH-dependent shift in its fluorescence excitation spectrum. Fluorescence emission (FEM) is slightly pH dependent with excitation (AEx) at 350-415 nm but highly pH dependent with AEX at 450 nm. Liposomes bearing a net negative charge bound rapidly to CV-1 cells and underwent endocytosis. One hour after liposome addition, high FEM with XEx at 413 nm and low FEM with XEX at 450 nm suggest that most cell-associated liposomes had been internalized and resided at a mean pH of zyxwvutsrqp -6.6. Collapse of cellular H+ gradients with NH4Cl or monensin treatment rapidly and reversibly increased FEM with XEX at 450 nm. Direct examination by fluorescence microscopy corroborates the fluorometric data on internalization; over time, FEM remained high with AEX at 350-405 nm but decreased with XEx at 450-490 nm, showing that all lipid vesicles were internalized within 40 min at 37 OC. Acidification of intracellular liposomes increased over 3 h, reaching a minimum value of approximately pH zyx 5.5. HPTS persisted within acidic cellular vesicles for 2-3 days, and cytoplasmic dye was observed infrequently, suggesting that liposome fusion with cellular membranes seldom occurs. Material delivered to the endocytic pathway via lipid vesicles labeled an assortment of intracellular organelles of varying motility and morphology, including dynamic tubular structures whose lumen is acidic. Liposomes have been used as model systems to study the biochemistry and biophysics of membrane processes, as probes of cellular function, and as carriers for cellular delivery of biologically active agents in vivo and in vitro. As model systems, they have contributed to the substantial progress made toward understanding membrane aspects of cellular (Hong et al., 1987; Meers et al., 1987) and viral fusion (White et al., 1980, 1982; Klappe et al., 1986; Nir et al., 1986), and the functional role of clathrin-associated components in recep- tor-mediated endocytosis (Chin et al., 1989). With regard to applications as carriers of macromolecules, liposomes con- taining chemotherapeutic agents are under evaluation for use against a variety of parasitic, fungal, and neoplastic diseases of humans (Mayhew zyxwvutsrq & Papahadjopoulos, 1983; Mayhew et al., 1984; Lopez-Berestein et al., 1985). In vitro, liposomes have been used for cytoplasmic delivery of labile or mem- brane-impermeant compounds in order to modify cell behavior (Wilson et al., 1979; Fraley et al., 1981; Reisine et al., 1986; Connor & Huang, 1986). Further development of liposomes for cell-biological and medical applications requires a detailed understanding of the mechanisms by which they interact with cells. Perhaps because of certain unique properties of liposomes, some early inves- tigations of liposome-cell interaction concluded that fusion of liposomes with the plasma membrane occurred with high frequency. Recent evidence suggests that liposomes composed of charged phospholipids or ligand-directed by means of 'This work was supported by Grants CA-35340 and CA-25526 from * Address correspondence to either author. *Department of Pharmaceutics, 539 Cooke Hall, University at Buf- '1 Cancer Research Institute, University of California. the National Institutes of Health. falo, SUNY. Electronic mail: rms@acsu.buffalo.bitnet (or .edu). Department of Pharmacology, University of California. surface-linked immunoglobulins may be endocytosed by cells (Machy et al., 1982; Heath et al., 1983; Huang et al., 1983; Straubinger et al., 1983; Truneh et al., 1983). Liposomes can enter cells through the coated pit/coated vesicle system (Straubinger et al., 1983) that is associated with internalization of many viruses or nutritional and signal molecules (Brown & Goldstein, 1979; Brown et al., 1983; Steinman et al., 1983; Mellman et al., 1986). It is not known currently how such encapsulated macromolecules as RNA (Wilson et al., 1979), DNA (Fraley et al., 1981), proteins (Gardas & Macpherson, 1979; McIntosh & Heath, 1982), or highly charged small molecules (Heath et al., 1983) escape the endocytic pathway and gain access to the cytoplasm functionally intact. In order to study the intracellular disposition of liposomes, our approach was to improve detection by light microscopy or fluorometry of the acidification of lipid vesicle aqueous contents (Hong et al., 1986), using hydroxypyrenetrisulfonate (HPTS),' as a fluorescence indicator for pH measurements in the physiological range. Fluorometry has been used to provide a quantitative assessment of liposome endocytosis by populations of cells, and fluorescence microscopy has been used to provide a qualitative view of the intra- and intercellular heterogeneity of liposome uptake. MATERIALS AND METHODS Reagents. HPTS was purchased from Molecular Probes (Eugene, OR) and did not require subsequent repurification. A 35 mM solution was prepared in 10 mM HEPES buffer, pH 7.4; NaCl was added to adjust the osmolality to 300 mOs/kg. Phosphatidylserine (from bovine brain) and phos- ' Abbreviations: CHOL, cholesterol; EDTA, ethylenediaminetetra- acetate; FEM, fluorescence emission intensity; HPTS, 8-hydroxy- zy 1,3,6- pyrenetrisulfonate; PBS, Dulbecco's phosphate-buffered saline; PBS/ CM, PBS with 0.4 zyxwv mM Ca2+ and 0.4 mM Mg2+; PC, phosphatidyl- choline; PS, phosphatidylserine; A , fluorescence excitation wavelength. 0006-2960/90/0429-4929$02.50/0 0 1990 American Chemical Society