Endocytosis and intracellular fate of liposomes using pyranine as a probe (original) (raw)
Related papers
Cell, 1983
We have compared the intracellular fate of several fluorescent probes and colloidal gold entrapped in negatively charged liposomes. Weakly acidic molecules (carboxyfluorescein) appear in the cytoplasm of CV-1 cells in 30 min; agents that raise lysosomal pH block this process. Highly charged molecules (calcein) and large molecules (FITC-dextran: 18 kd) remain confined to extra-or intracellular vesicles. Thin section electron micrographs show gold-containing liposomes bound to coated pits, in intracellular coated and uncoated vesicles, and in secondary lysosomes, including dense bodies. Free gold was not observed in the cytoplasm. We conclude that negatively charged liposomes are endocytosed and processed intracellularly by the coated vesicle pathway, and acidification of the endocytic vesicle, rather than liposome fusion, permits escape of certain molecules to the cytoplasm.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990
The interaction of liposomes with macrophage cells was monitored by a new fluorescence method (Hong, K., Straubinger, R.M. and Papahadjopoulos, D., J. Cell Biol. 103 (1986) 56a) that allows for the simultaneous monitoring of binding, endocytosis, acidification and leakage. Profound differences in uptake, cell surface-induced leakage and leakage subsequent to endocytosis were measured in liposomes of varying composition. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, I-IllS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of large unilamellar vesicles. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm) which have a complementary pH dependence in the range 5-9: the peak at 403 nm is maximal at low pH values while the peak at 450 nm is maximal at high pH values. The intra-and extracellular distribution of liposomes and their approximate pH was observed by fluorescence microscopy using appropriate excitation and barrier filters. The uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was monitored by spectrofluorometry via alterations in the fluorescence excitation maxima. The concentration of dye associated with cells was determined by measuring fluorescence at a pH independent point (413 nm). The average pH of ceil-associated dye was determined by normalizing peak fluorescene intensities (403 nm and 450 nm) to fluorescence at 413 nm and comparing these ratios to a standard curve. HPTS-containing liposomes bound to and were acidified by a cultured murine macrophage cell line (J774) with att/ 2 of 15-20 min. The acidification of liposomes exhibited biphasic kinetics and 50-80% of the liposomes reached an average pH lower than 6 within 2 h. A liposomal lipid marker exhibited a rate of uptake similar to HPTS, however the lipid component selectively accumulated in the cell; after an initial rapid release of liposome contents, 2.5-fold more lipid marker than liposomal contents remained associated with the cells after 5 h. Coating haptenated iiposomes with antibody protected liposomes from the initial release. The leakage of iiposomal contents was monitored by co-encapsulating I-IPTS and p-xylene-bis-pyridinium bromide, a fluorescence quencher, into liposomes. The time course of dilution of liposome contents, detected as an increase in HPTS fluorescence, was coincident with the acidification of HPTS. The rate and extent of uptake of neutral and negatively charged liposomes was similar; however, liposomes opsonized with antibody were incorporated at a higher rate (2.9-fold) and to a greater extent (3.4-fold). In addition, the rate and extent of incorporation of liposome encapsulated HPTS was dependent on temperature and the metabolic state of the cell, consistent with uptake of liposomes by endocytosis. The use of HlYFS allowed accurate and simultaneous quantitation of iiposome uptake, acidification, cell-induced leakage of liposomes, and regurgitation of liposome contents. In addition to cell-surface induced leakage, liposomes leaked extensively during endocytosis coincident with acidification; half of the cell-induced dilution of liposome contents was accounted for by leakage at the cell surface, while the remainder occurred coincident with acidification. Liposome contents labeled the aqueous space of endosomes and lysosomes and were regurgitated rapidly as liposomal lipid accumulated selectively. Opsonization of iiposomes, to induce Fc-mediated endocytosis, afforded protection to the initial dilution of liposome contents, but not the rate of leakage, after endocytosis. Implications of these studies for the use of liposomes as drug delivery vehicles are discussed.
Colloids and Surfaces B: Biointerfaces, 2007
In order to study mechanisms involved in liposome-cell interaction, this work attempted to assess the influence of vesicle composition on the delivery of liposomal content to Hela cells. In particular, to evaluate pH-sensitive properties and cell interaction of the prepared liposomes, the lipid formulations contained cholesterol (Chol) and they were varied by using phosphatidylcholines with different purity degree: soy lecithin (SL; 80% phosphatidylcholine), a commercial mixture of soy phosphatidylcholine (P90; 90% phosphatidylcholine) or dipalmitoylphosphatidylcholine (DPPC; 99% of purity). A second series of liposomes also contained stearylamine (SA). Dehydration-rehydration vesicles (DRV) were prepared and then sonicated to decrease vesicle size. Vesicle-cell interactions and liposomal uptake were examined by fluorescence microscopy using carboxyfluorescein (CF) and phosphatidylethanolamine-dioleoyl-sulforhodamine B (Rho-PE) as fluorescent markers. Fluorescence dequenching assay was used to study the influence of pH on CF release from the liposomal formulations. Liposome adhesion on the cell surface and internalization were strongly dependent on vesicle bilayer composition. SA vesicles were not endocytosed. DPPC/Chol liposomes were endocytosed but did not release their fluorescent content into the cytosol. SL/Chol and P90/Chol formulations displayed a diffuse cytoplasmic fluorescence of liposomal marker. (A.M. Fadda).
Biophysical studies and intracellular destabilization of pH-sensitive liposomes
Lipids, 2000
We examined changes in membrane properties upon acidification of dioleoylphosphatidylethanolamine/cholesterylhemisuccinate liposomes and evaluated their potential to deliver entrapped tracers in cultured macrophages. Membrane permeability was determined by the release of entrapped calcein or hydroxypyrene-1,3,6-trisulfonic acid (HPTS)-p-x ylene-bis-pyridinium bromide (DPX); membrane fusion, by measuring the change in size of the liposomes and the dequenching of octadecylrhodamine-B fluorescence; and change in lipid organization, by 3 1 P nuclear magnetic resonance spectroscopy. Measurement of cell-associated fluorescence and confocal microscopy examination were made on cells incubated with liposomes loaded with HPTS or HPTS-DPX. The biophysical studies showed (i) a lipid reorganization from bilayer to hexagonal phase progressing from pH 8.0 to 5.0, (ii) a membrane permeabilization for pH <6.5, (iii) an increase in the mean diameter of liposomes for pH <6.0, and (iv) a mixing of liposome membranes for pH <5.7. The cellular studies showed (i) an uptake of the liposomes that were brought from pH 7.5-7.0 to 6.5-6.0 and (ii) a release of ~15% of the endocytosed marker associated with its partial release from the vesicles (diffuse localization). We conclude that the permeabilization and fusion of pH-sensitive liposomes occur as a consequence of a progressive lipid reorganization upon acidification. These changes may develop intracellularly after phagocytosis and allow for the release of the liposome content in endosomes associated with a redistribution in the cytosol.
On the mechanisms of internalization and intracellular delivery mediated by pH-sensitive liposomes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2001
We investigated the molecular mechanisms by which pH-sensitive liposomes surpass the cytoplasmic and endosomal membranes to deliver their aqueous contents into the cytoplasm. Various liposome formulations were evaluated for their efficacy to mediate intracellular delivery of encapsulated material, including a novel sterically stabilized pH-sensitive formulation ((DOPE:CHEMS:DSPE-PEG 2000 (6:4:0.3)) that was previously developed in our laboratories. In an attempt to fully characterize the nature of liposome^cell interactions different approaches based on a dual-labeling fluorescence assay were used. Our results indicate that the efficacy of interaction of pH-sensitive liposomes, both plain and sterically stabilized, with cells is strongly determined by the inclusion of DOPE in their composition, independently of the type of the amphiphilic stabilizer used. In fact, DOPE-containing liposomes shown to be non-pH sensitive by biophysical assays, mediated cytoplasmic delivery of their contents as efficiently as well known pH-sensitive formulations (e.g. DOPE:CHEMS). However, among the different formulations studied, DOPE:CHEMS liposomes were those exhibiting the highest extent of cell association. Moreover, our results with cells pretreated with metabolic inhibitors or lysosomotropic agents clearly indicate that DOPE-containing liposomes are internalized essentially by endocytosis and that acidification of the endosomes is not the only mechanism involved in the destabilization of the liposomes inside the cell. ß
Springer eBooks, 1984
We have compared the intracellular fate of several fluorescent probes and colloidal gold entrapped in negatively charged liposomes. Weakly acidic molecules (carboxyfluorescein) appear in the cytoplasm of CV-1 cells in 30 min; agents that raise lysosomal pH block this process. Highly charged molecules (calcein) and large molecules (FITC-dextran: 18 kd) remain confined to extra-or intracellular vesicles. Thin section electron micrographs show gold-containing liposomes bound to coated pits, in intracellular coated and uncoated vesicles, and in secondary lysosomes, including dense bodies. Free gold was not observed in the cytoplasm. We conclude that negatively charged liposomes are endocytosed and processed intracellularly by the coated vesicle pathway, and acidification of the endocytic vesicle, rather than liposome fusion, permits escape of certain molecules to the cytoplasm.
Fusion of cationic liposomes with mammalian cells occurs after endocytosis
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1995
The interaction of cationic liposomes prepared using either dioleoyltrimethylammonium propane (DOTAP) or 3~8-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-CHOL) with model membranes and with cultured mammalian cells was examined using an assay developed for monitoring virus-cell fusion (Stegmann et al. (1993) Biochemistry 32, 11330-11337). Lipid mixing between cationic liposomes and liposomes composed of DOPE/dioleoylphosphatidyiglycerol (DOPG) or dioleoylphosphatidylcholine (DOPC)/DOPG was insensitive to pH in the range of pH 4.5-7.0 and was not affected by sodium chloride concentration in the range of 0-150 mM. Lipid mixing was dependent on dioleoylphosphatidylethanolamine (DOPE), since cationic liposomes prepared using dioleoylphosphatidylcholine (DOPC) were incapable of lipid mixing with DOPC/DOPG liposomes. The interaction of cationic liposomes with Hep G-2 and CHO D-cells was also studied. For both cell types, liposome-cell lipid mixing was rapid at 37 ° C, beginning within minutes and continuing for up to 1 hour after uptake. The extent of lipid mixing was decreased at 15 ° C, especially at later (> 20 min) time points. This suggests that at least part of the observed lipid mixing occurred after reaching cellular lysosomes. No lipid mixing was seen at 4 ° C. Monensin inhibited lipid mixing between cationic liposomes and the cells, despite having no effect on liposome uptake. Inhibition of endocytic uptake of liposomes, either by incubation in hypertonic media or by depletion of cellular ATP with sodium azide and 2-deoxyglucose abolished liposome-cell fusion in both cell types. These data demonstrate that binding to the cell surface is insufficient for cationic liposome-cell fusion and that uptake into the endocytic pathway is required for fusion to occur.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1992
We investJpted the interaction of liposomes of different surface properties with two mammalian tell lines, CVI, mt African j~en monkey kidney cell line, and J774, n murine macrophnge.Uke cell line. Cell surface binding mid endoeytmls of liposomes were quantified by fluorometry, using the iiposonte-encapsuiated pH.set~sitive fJnecesc~ dye, pyranine, and the lipid marker rbedamine.PE. The IJpecome uptake was dependent i'~th on tile snef~ properties of the lipesomes and on the cell line. Neptivety charsed pbospholJpids inc~perated into e88 Idmepi~ tJdylcholine (l~)/cholestscol (C) (2:1) Upocomes were recognized by the two cell lines to ~ extents depending on the lipid headlpmup and its dmrge density in the lipocome bUayer, lnctusion of 9% ~,bosphaJd34set'~ (PS), phosphatidyllllycerol (PG), or phosphatidic ~,.i3 (PA) promoted the uptake by CV1 cells more than 20-fold. Increasing the content of these negatively eharled lipids beyond 9% did not further enhance the uptake. In 9% moncoisiopngikeide Gin, phesphatidyJ~uosltoJ (Pi), or phospha~uolamine conjupted to pe~etk~se glycol) (PEG-PE) did not promote the uptake. Inclusion of 9% PS, PG, PA or PI in PC/C ligecom~; did let enhsnce the uptake by J774 cells, bat a drastic enhancement was observed when ittaensit~, concentrations of these anionic lipids were incorporated in the lipeseme bilnyer. At k~st 50% IS, I~, or P! was needed to reach t~ level of uptake seen with CV! cells. The uptake of Upecomes ¢ontainiq ~ PS by J774 cells wgs inbibitsd by pely.oniom which are the competing Iillaads for ~'sveager receptors, bat the al~tke by ~'1 was not ir~hibited. Di~qrcot mechanisms of lilmcome ul~dte by these two cell lines ore mllgeSted from tke different patterns of apta~ mid competition w~th rations pely-onione. The differences observed in the uptake rate of lilmcomes with dilrmt lipid compositions seemed to be primarily due to the differenees Jn the binding I~tween lil~sOmes and ¢¢.11 components. The in v~e interaction of various lipesomes with these cell lines, especially CYl ce~ shows significant similarities to the in vi~ clearance rates of the Ijposomes.
Journal of Biomedical Optics, 2005
Liposomes are known to be taken up by the liver cells after intravenous injection. Among the few techniques available to follow this process in vivo are perturbed angular correlation spectroscopy, nuclear magnetic resonance spectroscopy, and scintigraphy. The study of the intracellular pathways and liposomal localization in the different liver cells requires sacrifice of the animals, cells separation, and electronic microscopy. In the acidic intracellular compartments, the in situ rate of release of liposomes remains poorly understood. We present a new method to follow the in situ and in vivo uptake of liposomes using a fluorescent pH-sensitive probe 5,6carboxyfluorescein (5,6-CF). 5,6-CF is encapsulated in liposomes at high concentration (100 mM) to quench its fluorescence. After laparotomy, liposomes are injected into the penile vein of Wistar rats. Fluorescence images of the liver and the skin are recorded during 90 min and the fluorescence intensity ratio is calculated. Ratio kinetics show different profiles depending on the liposomal formulation. The calculated intracellular liver pH values are, respectively, 4.5 to 5.0 and 6.0 to 6.5 for DSPC/chol and DMPC liposomes. After sacrifice and flush with a cold saline solution, the pH of the intracellular site of the liver (ex vivo) is found to be 4.5 to 5.0. This value can be explained by an uptake of liposomes by the liver cells and subsequent localization into the acidic compartment. An intracellular event such as dye release of a drug carrier (liposomes loaded with a fluorescent dye) can be monitored by pH fluorescence imaging and spectroscopy in vivo and in situ.
Interactions between pH-sensitive liposomes and model membranes
Biophysical Chemistry, 2003
The structure and dynamics of two different pH-sensitive liposome systems were investigated by means of cryotransmission electron microscopy and different photophysical techniques. Both systems consisted of dioleoylphosphatidylethanolamine (DOPE) and contained either oleic acid (OA) or a novel acid-labile polyethylene glycol-conjugated lipid (DHCho-MPEG5000) as stabiliser. Proton induced leakage, lipid mixing and structural changes were studied in the absence and presence of EPC liposomes, as well as in the presence of liposomes designed to model the endosome membrane. Neither DHCho-MPEG5000-nor OA-stabilised liposomes showed any tendency for fusion with pure EPC liposomes or endosome-like liposomes composed of EPCyDOPEySMyCho (40y20y6y34 mol.%). Our investigations showed, however, that incorporation of lipids from the pH-sensitive liposomes into the endosome membrane may lead to increased permeability and formation of non-lamellar structures. Taken together the results suggest that the observed ability of DOPE-containing liposomes to mediate cytoplasmic delivery of hydrophilic molecules cannot be explained by a mechanism based on a direct, and non-leaky, fusion between the liposome and endosome membranes. A mechanism involving destabilisation of the endosome membrane due to incorporation of DOPE, seems more plausible. ᮊ