Incorporation of cholesterol into high density lipoprotein recombinants.

Apo-A-1, the principal apoprotein of high density lipoprotein, was incubated with cholesterol containing liposomes of dimyristoyl lecithin, lecithin from high density lipoprotein or sphingomyelin. Conditions were chosen to give 100% conversion of cholesterol-free liposomes into recombinants which were isolated by density gradient ultracentrifugation. For all phospholipids, there was a progressive decrease in incorporation of lipid into recombinants with increasing cholesterol/phospholipid ratio. The cholesterol/phospholipid ratio of recombinants was ～ 45% of unreacted liposomes, for all initial cholesterol/phospholipid ratios. The reduced cholesterol content suggests exclusion of cholesterol from a fraction of recombinant phospholipid, probably a boundary layer in contact with apo A-1.     

Photosensitive liposomes as 'cages' for laser-triggered solute delivery: the effect of bilayer cholesterol on kinetics of solute release

Liposomes containing acyl chains incorporating azobenzene chromophores have been investigated as potential 'caging' agents for fast solute release. On photolysis, trapped marker dye can be released from gel-phase liposomes within milliseconds. Solute release is markedly sensitive to the presence of cholesterol in the bilayer. Phospholipids bearing one saturated acyl chain and an azobenzene-substituted chain are ineffective as sensitisers unless cholesterol is present, while doubly substituted phospholipids sensitise release in its absence. Cholesterol markedly affects the temperature profile of solute release depending on the host phospholipid chain length. Solute release is not seen for lipid hosts with unsaturated acyl chains.     

Interaction of cholesterol, phospholipid and apoprotein in high density lipoprotein recombinants.

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of 3H cholesterol/14C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27 degrees C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1, or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. 160 X 55 A. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Interaction of cholesterol,phospholipid and apoprotein in high density lipoprotein recombinants

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of ³H cholesterol/¹⁴C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27°C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1 or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. $\text{160 × 55}\text{A}\text{?}$. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Effect of cholesterol on the uptake and intracellular degradation of liposomes by liver and spleen; a combined biochemical and gamma-ray perturbed angular correlation study

We investigated the effect of cholesterol on the uptake and intracellular degradation of liposomes by rat liver and spleen macrophages. Multilamellar vesicles (MLV) consisting of distearoylphosphatidylcholine/phosphatidylserine (molar ratio 9:1) or distearoylphosphatidylcholine/cholesterol/phosphatidylserine (molar ratio 4:5:1) were labeled with [3H]cholesteryl hexadecyl ether and/or cholesteryl [14C]oleate. After i.v. injection the cholesterol-containing liposomes were eliminated less rapidly from the bloodstream and taken up to a lesser extent by the liver (macrophages) than the cholesterol-free liposomes. Assessment of the 3H/14C ratios in liver and spleen cells revealed that the cholesterol-containing liposomes are substantially more resistant towards intracellular degradation than the cholesterol-free liposomes. These results could be confirmed by measuring the release of 111In from liposomes after uptake by liver and spleen by means of gamma-ray perturbed angular correlation spectroscopy. Experiments with cultured Kupffer cells in monolayer also revealed that incorporation of cholesterol results in a decrease of the uptake and an increase of the intracellular stability of cholesteryl [14C]oleate-labeled liposomes. Finally, incubation of both types of liposomes with lysosomal fractions prepared from rat liver demonstrated a difference in susceptibility to lysosomal degradation: the cholesterol-free vesicles were much more sensitive to lysosomal esterase than the cholesterol-containing liposomes. These results may be relevant to the application of liposomes as a drug carrier system to liver and spleen (macrophages).     

Differential effect of triiodothyronine and thyroxine on liposomes containing cholesterol: Physiological speculations

The effect of thyroid hormones on the steadystate fluorescence polarization and on the release of the liposomal content was analyzed in liposomes composed of egg phosphatidylcholine and egg phosphatidyl choline: cholesterol in different molar ratios. Depending on liposome cholesterol composition, a dual effect of triiodothyronine was found. The fluorescence polarization of 1,6 diphenyl 1,3,5 hexatriene or 1-(4-trimethylaminophenyl) 6 phenyl-1, 3, 5 hexatriene decreased by the addition of the hormone when cholesterol content was in the range from 0 to 30 moles %, while it increased with cholesterol from 30 to 50 moles %. In the release experiments, the effect of triiodothyronine was also biphasie; the leakage was the highest at 0% and 50% and the lowest at 30 moles % of cholesterol. On the contrary, thyroxine was without effect on liposomes containing cholesterol from 30 to 50 mol %. This fact correlated with a lower incorporation of thyroxine, compared with that of triiodothyronine in liposomes containing up to 30 moles % of cholesterol.The fact that the above differential incorporation of thyroid hormones was also observed at physiological concentration and that most of the mammalian membrane cells have more than 25 moles % of cholesterol have for physiological implications to the observations reported here.     

Phosphatidylcholine-rich acceptors, but not native HDL or its apolipoproteins, mobilize cholesterol from cholesterol-rich insoluble components of human atherosclerotic plaques

To examine the potential of high density lipoproteins (HDL) to ameliorate atherosclerotic plaques in vivo, we examined the ability of native HDL, lipid-free HDL apolipoproteins (apo HDL), cholesterol-free discoidal reconstituted HDL (R-HDL) comprised of apo HDL and phosphatidylcholine (PC) and PC liposomes to release cholesterol from cholesterol-rich insoluble components of plaques (ICP) isolated from atherosclerotic human aorta. Isolated ICP had a free cholesterol (FC) to phospholipid (PL) mass ratio (0.8-3.1) and a sphingomyelin (SPM) to PC mass ratio (1.2-4.2) that exceeded those of plasma membranes of cultured cells. Surprisingly, native HDL and its apolipoproteins were not able to release cholesterol from ICP. However, R-HDL and PC liposomes were effectively released cholesterol from ICP. The release of ICP cholesterol by R-HDL was dose-dependent and accompanied by the transfer of > 8 x more PC in the reverse direction (i.e., from R-HDL to ICP), resulting in a marked enrichment of ICP with PC. Compared to R-HDL, PC liposomes were significantly less effective in releasing cholesterol from ICP but were somewhat more effective in enriching ICP with PC. Native HDL was minimally effective in enriching ICP with PC, but became effective after prior in vitro enrichment of HDL with PC from multilamellar PC liposomes. The enrichment of ICP with PC resulted in the dissolution of cholesterol crystals on ICP and allowed the removal of ICP cholesterol by apo HDL and plasma. Our study revealed that the removal of cholesterol from ICP in vivo will be possible through a change in the level, composition, and physical state of ICP lipids mediated by PC-enriched HDL.     

The effect of cholesterol in the liposome bilayer on the stabilization of incorporated Retinol

The effect of cholesterol in the liposome bilayer on the stability of incorporated retinol was studied. Retinol was incorporated into liposomes containing soybean phosphatidylcholine (PC) and cholesterol (CH) at various ratios, and the liposomes were prepared as multilamellar vesicles by the dehydration-rehydration method. Retinol readily incorporated into liposomes at a ratio of 0.01:1 (w/w) retinol:lipid, with over 94.52% being incorporated in all conditions studied. The incorporation efficiency of retinol increased slightly with increasing CH content in the liposome and with increasing pH of the hydration buffer. Average particle size increased as the CH content increased, and mean particle sizes at pH 5, 7, and 9 were 30.27, 89.53, and 41.42 microm, respectively. The time course of retinol degradation in aqueous solution in liposomes with various ratios of PC to CH was determined under a variety of pH conditions (pH 5, 7, and 9), and temperatures (4, 25, 37, and 50 degrees C). The stability of incorporated retinol was enhanced by increasing the CH content. At pH 7.0 and 4 degrees C, for example, 90.17% of the retinol in liposomes containing 50:50 (PC:CH) remained after 10 days of storage, whereas 51.46% remained at 100:0 (PC:CH). These results indicate that CH in liposomes greatly increases the incorporation efficiency of retinol and the stability of incorporated retinol.     

Interaction of digitonin and its analogs with membrane cholesterol

The interaction of digitonin with membrane cholesterol was studied by using various digitonin analogs, and radioactive desglucodigitonin. The following results were obtained concerning the effect of digitonin on erythrocytes, granulocytes and liposomes. Digitonin and its analogs showed activity to induce hemolysis, granulocyte activation and liposomal membrane damage. The activity was affected by change of the carbohydrate residue of the molecule; the order of hemolytic activity was digitonin greater than or equal to desglucodigitonin much greater than glucosyl-galactosyl-digitogenin greater than galactosyl-digitogenin, digitogenin. The relative activities of these compounds to induce granulocyte activation and liposomal membrane damage were similar to those observed in the hemolysis. [3H]Desglucodigitonin could bind to cholesterol in liposomes. The binding was stoichiometric and the ratio of desglucodigitonin bound to liposomes/cholesterol in liposomes was close to 1, irrespective of the cholesterol content in liposome. Damage to liposomes was, however, induced by desglucodigitonin only when they contained more than 0.2 molar ratio of cholesterol to phospholipid. Addition of digitonin as well as desglucodigitonin to preformed liposomes deprived of cholesterol affected the anisotropic molecular motion of spin-labeled phosphatidylcholine incorporated into the liposomes, suggesting that the molecules could be inserted into the lipid bilayer free of cholesterol. Molecules of desglucodigitonin in the lipid phase may, however, be equilibrated with those in the aqueous phase, unless they form a complex with cholesterol, since no appreciable amount of [3H]desglucodigitonin could be detected in the liposome fraction after separation by column chromatography. Digitonin decreased the order parameter of spin-labeled phosphatidylcholine when liposomes contained equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competitive carotenoid and cholesterol incorporation into liposomes: effects on membrane phase transition, fluidity, polarity and anisotropy

Pure 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) or mixed DPPC:1,2-dipalmitoyl phosphatidyletanolamine (DPPE):1,2-dipalmitoyl diphosphatidylserine (DPPS) (17:5:3) liposomes were incorporated with 5 mol% dietary carotenoids (beta-carotene, lutein and zeaxanthin) or with cholesterol (16 and 48 mol%) in the absence or presence of 15 mol% carotenoids, respectively. The carotenoid incorporation yields ranged from 0.42 in pure to 0.72 in mixed phospholipid liposomes. They decreased significantly, from 3 to 14%, in the corresponding cholesterol-doped liposomes, respectively. Highest incorporation yields were achieved by zeaxanthin and lutein in phospholipid liposomes while in cholesterol-containing liposomes, lutein was highest incorporated. The effects on membrane structure and dynamics were determined by differential scanning calorimetry, steady-state fluorescence and anisotropy measurements. Polar carotenoids and cholesterol cause similar, dose-dependent effects: ordering and rigidification revealed by broadening of the transition peak, and increase of anisotropy. Membrane hydrophobicity is determined by cholesterol content and carotenoid polarity. In cholesterol-doped liposomes, beta-carotene is less incorporated than in cholesterol-free liposomes. Our observations suggest effects of carotenoids, even at much lower effective concentrations than cholesterol (8 to 80-fold), on membrane structure and dynamics. Although they are minor constituents of animal membranes, carotenoids may act as modulators of membrane phase transition, fluidity, polarity and permeability, and therefore, can influence the membrane physiology and pathology.     

Membrane fluidity changes in goat sperm induced by cholesterol depletion using beta-cyclodextrin

Cholesterol efflux from membranes promotes acrosome reaction in goat spermatozoa. In 1 h of incubation of sperm in the presence of beta-cyclodextrin (beta CD), all the interchangeable cholesterol is desorbed from sperm membranes, although acrosome reaction is fully accomplished only after 3-4 h of incubation, as previously published. In the present paper we investigate the effect of cholesterol removal from mature goat spermatozoa on the overall membrane "fluidity" of live cell membranes and of liposomes from sperm lipid extracts. Using steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), we studied the average thermotropic behaviour of membrane lipids, after incubation of live sperm for 1 h in BSA-free medium with the presence/absence of 8 mM beta-cyclodextrin, as a cholesterol acceptor. Unimodal and bimodal theoretical sigmoids fitted best to the experimental thermotropic profiles of liposomes and whole cells, respectively. In the case of whole sperm, two phase transitions, attributable to different lipid domains, were clearly separated by using the fitting parameters. After cholesterol removal, important changes in the relative anisotropy range of the two transitions were found, indicating an increase in the "fluidity" of some of the lipid microdomains of sperm membranes. These changes in sperm lipid dynamics are produced before the onset of sperm acrosome reaction.     

In vitro modification of cholesterol content of rat liver microsomes. Effects upon membrane 'fluidity' and activities of glucose-6-phosphatase and fatty acid desaturation systems

The cholesterol content of rat liver microsomal membranes was modified in vitro by incubating microsomes and cytosol with liposomes prepared by sonication of microsomal lipids and cholesterol. In this way, the cholesterol to phospholipid molar ratio was increased from 0.11-0.13 in untreated microsomes to a maximal of 0.8 in treated ones. Cholesterol incorporation in microsomes produced an increase in the diphenyl-hexatriene steady-state fluorescence anisotropy and a decrease in the efficiency of pyrene-excimer formation which indicated a decrease in the rotational and translational mobility, respectively, of these probes in the membranes lipid phase. Cholesterol incorporation in microsomes did not affect significantly the glucose-6-phosphatase activity in 0.1% Triton X-100 totally disrupted microsomes, but diminished the glucose-6-phosphatase activity of 'intact' microsomes. This indicates that possibly the glucose 6-phosphate translocation across the microsomal membrane is impeded by an increase in the membrane apparent 'microviscosity'. Cholesterol incorporation in microsomes decreased NADH-cytochrome c reductase without affecting NADH-ferricyanide reductase activity. The delta 9 desaturation reaction rate was enhanced by cholesterol incorporation at low but not at high palmitic acid substrate concentration. delta 5 and delta 6 desaturase reaction-rates were increased both at low and high fatty acid substrate concentrations. These results suggest that a mechanism involving fatty acid desaturase enzymes, might exist to self-regulate the microsomal membrane lipid phase 'fluidity' in the rat liver.     

Lipoproteins do not influence cholesterol synthesis in freshly isolated rat hepatocytes, cautionary note

1. Suspensions of freshly isolated rat hepatocytes were used to study the effects of native and derivatized lipoproteins on the rate of cholesterogenesis. 2. Short-term incubation of the hepatocytes with a variety of lipoproteins failed to modify the rate of cholesterol synthesis as determined by the incorporation of tritium from tritiated water into cholesterol after separation from other lipids by thin-layer chromatography. 3. Neither an increase in the cholesterol content of the particles (beta-very-low-density lipoproteins) nor derivatization of the lipoproteins (lactosylated-low-density lipoproteins or high-density lipoproteins associated with a tris-galactoside-terminated cholesterol derivative) nor cholesterol-containing liposomes were effective in this respect. 4. Whether this behaviour represents an artefact of the isolated hepatocyte preparation is unknown yet.     

Effects of cholesterol on lipid organization in human erythrocyte membrane

The molar ratio of cholesterol to phospholipid (C/P) in human erythrocyte membrane is modified by incubating the cells with liposomes of various C/P ratios. The observed increase in cell surface area may be accounted for by the addition of cholesterol molecules. Fusion between liposomes and cells or attachment of liposomes to cells is not a significant factor in the alteration of C/P ratio. Onset temperatures for lipid phase separation in modified membranes are measured by electron diffraction. The onset temperature increases with decreasing C/P ration from 2 degrees C at C/P = 0.95 to 20 degrees C at C/P = 0.5. Redistribution of intramembrane particles is observed in membranes freeze-quenched from temperatures below the onset temperature. The heterogeneous distribution of intramembrane particles below the onset temperature suggests phase separation of lipid, with concomitant segregation of intramembrane protein into domains, even in the presence of an intact spectrin network.     

Membrane fluidity changes in goat sperm induced by cholesterol depletion using beta-cyclodextrin

Cholesterol efflux from membranes promotes acrosome reaction in goat spermatozoa. In 1 h of incubation of sperm in the presence of beta-cyclodextrin (βCD), all the interchangeable cholesterol is desorbed from sperm membranes, although acrosome reaction is fully accomplished only after 3-4 h of incubation, as previously published. In the present paper we investigate the effect of cholesterol removal from mature goat spermatozoa on the overall membrane “fluidity” of live cell membranes and of liposomes from sperm lipid extracts. Using steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), we studied the average thermotropic behaviour of membrane lipids, after incubation of live sperm for 1 h in BSA-free medium with the presence/absence of 8 mM β-cyclodextrin, as a cholesterol acceptor. Unimodal and bimodal theoretical sigmoids fitted best to the experimental thermotropic profiles of liposomes and whole cells, respectively. In the case of whole sperm, two phase transitions, attributable to different lipid domains, were clearly separated by using the fitting parameters. After cholesterol removal, important changes in the relative anisotropy range of the two transitions were found, indicating an increase in the “fluidity” of some of the lipid microdomains of sperm membranes. These changes in sperm lipid dynamics are produced before the onset of sperm acrosome reaction.     

Preparation of liposomes containing lysosomal enzymes for therapeutic use

The preparation of fused materials using liposomes has been examined for several decades as a tool for the stabilization of heterogeneous enzymes. We investigated the liposomal encapsulation of lysosomal enzymes extracted from Saccharomyces cerevisiae. Liposomes were formed with L-α-phosphatidylcholine from egg yolk and cholesterol. To encapsulate whole lysosomal enzymes in liposomes made with and without cholesterol, L-α-phosphatidylcholine and cholesterol were added to chloroform at a ratio of 10:0 (L-α-phosphatidylcholine:cholesterol) and then evaporated for 10 min at 4°C. The residue after evaporation was mixed with lysosomal enzymes at the same ratio and then vortexed for 1 min and sonicated for 5 sec to encapsulate the enzymes. Liposome-encapsulated lysosomal enzymes were created using various amounts of lysosomal enzymes and cholesterol. The results indicated that the optimal encapsulation conditions were lipid:cholesterol ratios of 7:3 and 8:2. Liposome formation was confirmed by TEM imaging. After 1 day, two types of liposomes released small amounts of lysosomal enzymes. However, after 6 days, liposomes formed from mixtures of lipid and cholesterol did not exhibit any changes, whereas liposomes formed from only lipids released high amounts of lysosomal enzymes. Lysosomal enzymes encapsulated in liposomes have potential as important drug delivery carriers, as liposomes are able to control drug release and bioavailability.     

Lipid peroxidation contributes to age-related membrane rigidity

The aim of this work was to assess the relative contributions of lipid peroxidation and cholesterol content to the increase in membrane rigidity observed during senescence. Membrane fluidity was manipulated through exposure to peroxidized or cholesterol-loaded liposomes. Small unilamella liposomes were prepared and either peroxidized by Fe⁺⁺-ADP-ascorbic acid or loaded with cholesterol. After incorporation of the liposomes into rat liver microsomal membranes, membrane fluidity was quantitated by measuring changes in polarization. Membranes exhibited a greater sensitivity to peroxidation than cholesterol in that incorporation of peroxidized liposomes induced microsomal membrane rigidity substantially more than did cholesterol-loaded liposomes. Thus it is proposed, based on data from the present and earlier studies, that membrane fluidity can be modulated readily by lipid peroxidation of membrane phospholipids, irrespective of the influences of cholesterol. These results support the proposal that alterations of lipid structure are more potent and effective than compositional changes in cholesterol in inducing age-related increases in membrane rigidity.     

Complete exchange of viral cholesterol.

The exchange of the cholesterol in the membranes of two enveloped viruses, Sindbis virus and vesicular stomatitis virus, with cholesterol present in lipid vesicles and in serum was measured. Biosynthetically labeled viral cholesterol underwent spontaneous and complete transfer to both lipid vesicles and to serum. The rate with which and the extent to which this process occurred were very similar for these two viruses. During incubation with lipid vesicles in excess, half of the viral cholesterol underwent transfer in approximately 4 h and more than 90% underwent transfer in 24h at 37 degrees C. Similar rates and extents of movement of viral cholesterol were observed when incubations were carried out with vesicles which contained cholesterol and phospholipid in the same molar ratio as in the virus or with egg lecithin vesicles which contained no cholesterol. When labeled cholesterol was present initially in the lipid vesicles, movement of cholesterol from the vesicles to the virus was observed. One implication of the fact that viral cholesterol undergoes extensive exchange with serum cholesterol is that cellular cholesterol is in equilibrium with that in the extracellular fluid.     

The effect of a water-soluble tris-galactoside terminated cholesterol derivative on the in vivo fate of small unilamellar vesicles in rats

When the water-soluble cholesterol derivative, N-[tris [(beta-D-galactopyranosyloxy)methyl]methyl]-N alpha-[4-(5-cholesten-3 beta-yloxy)succinyl]glycinamide (tris-gal-chol) (Kempen et al. (1984) J. Medicin. Chem. 27, 1306-1312) is added as an aqueous micellar solution to a dispersion of small unilamellar phospholipid vesicles it rapidly associates with the vesicles, without causing significant leakage of liposome contents. Incorporation of 10 mol% tris-gal-chol in the liposomal membrane caused a substantial increase in the rate and extent of rat liver uptake and a shift in intrahepatic distribution of an intravenously administered dose of liposomes. For neutral liposomes composed of equimolar amounts of cholesterol and sphingomyelin incorporation of tris-gal-chol led to a 7-fold increase in total liver uptake, which was mainly accounted for by an increase in uptake by the Kupffer cells (12-fold) and by only a small increase in uptake by the hepatocytes (1.4-fold). The increased liver uptake is blocked by preinjection of N-acetyl-D-galactosamine and not affected by preinjection of N-acetyl-D-glucosamine. This indicates that the increased interaction of liposomes as a result of tris-gal-chol incorporation is mediated by galactose-specific recognition sites on both Kupffer cells and hepatocytes. Targeting of liposomes to the asialoglycoprotein receptor of the hepatocytes is thus frustrated by the highly active galactose-specific receptor on Kupffer cells. Comparable results on lactosylceramide incorporation into liposomes were recently reported by us (Spanjer et al. (1984) Biochim. Biophys. Acta 774, 49-55).