Modification of the lipid composition of neuroblastoma C1300 cells using liposomes

An essential increase in the total cholesterol content with a significant growth in the level of its esters was observed in neuroblastoma C1300 cells differentiated by means of 5'-bromodesoxyuridine and incubated for 1 h with lecithin-cholesterol liposomes (1:1 mol/mol). These cells also displayed an increased amount of polyunsaturated fatty acids in the composition of lecithin molecules and the appearance of lysolicethin as well. Incubation of the cells with lecithin liposomes was accompanied by a decrease in the content of cholincholesterol in the cells. In this case the level of cholesterol esters decreased to a greater extent than the level of free cholesterol. In lecithin molecules of these cells there occurred a relative increase of saturated fatty acids. The cells can retain viability and compensate changes caused by cholesterol excess during their incubation with lecithin-cholesterol liposome for 60-90 min. A longer incubation with liposomes results in a sharp drop of cell viability.     

Transmembrane lipid transport between lecithin liposomes and neuroblastoma C1300 N18 cells

The method of double isotopic labels was used to study dynamics of lipid metabolism between neuroblastoma C 1300 N 18 A 1 cells and lecithin liposomes which contained 4.5-5 mumol of lecithin in 1 ml of the suspension. The cell lipids were labelled by radioactive carbon and cultivated on the medium with [1-14C] sodium acetate, phosphatidylcholine of liposomes was labelled by tritium. It is shown that 15-30 min long incubation with liposomes causes a sharp decrease of the cholesterol esters amount with a simultaneous fall of the free cholesterol level. The total content of phospholipids in this case remains unchanged though there occurs the noticeable exchange of labelled phospholipids between cells and liposomes. The cholesterol content in the plasma membranes of cells lowers sharply. The neuroblastoma cells are able to compensate arising changes in the cholesterol level for 45-60 min after which they progressively die. 90 min later only an insignificant part of the population (about 10% of cells) is retained.     

Difference in microviscosity induced by different cholesterol levels in the surface membrane lipid layer of normal lymphocytes and malignant lymphoma cells

Microviscosity ($\text{}\text{?}$gh) in the surface membrane lipid layer of normal lymphocytes and malignant lymphoma cells, and in liposomes prepared from their lipid extracts, was determined with the aid of the fluorescence polarization properties of 1,6-diphenyl 1,3,5-hextriene embedded in it. The $\text{}\text{?}$gh values, both in intact cells and in the liposomes, are distinctively greater for normal lymphocytes than for the lymphoma cells, whereas the fusion activation energy in both types of cells and liposomes is 8 ± 0.5 kcal/mol. Determination of cholesterol revealed that its relative amount in a lymphoma cell is about half of that of a normal lymphocyte, a difference that may account for the above difference in fluidity. This thesis is supported by the observed changes in $\text{}\text{?}$gh, which follow artificial changes in cholesterol contents in the surface membrane of both cell types. Introduction of exogeneous cholesterol into the cell surface membranes was performed with lecithin-cholesterol (1:1) liposomes, and in lymphoma cells resulted in an increase of $\text{}\text{?}$gh to a level of normal lymphocytes. Extraction of native cholesterol from the cell surface membranes was carried out with lecithin liposomes, and in normal lymphocytes results in a decrease of $\text{}\text{?}$gh to a value similar to that of lymphoma cells. The induced changes in cholesterol contents are practically reversible for both cell types. By virtue of controlling the microviscosity of lipid layers, the level of cholesterol in cell surface membranes may play an important role in determining biological activities of normal and malignant cells.     

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.     

Encapsulation of Chicken Egg Yolk Immunoglobulin G (IgY) by Liposomes

Encapsulation of antibodies isolated from chicken egg yolk (IgY) in egg lecithin/cholesterol liposomes was attempted. IgY was successfully encapsulated into the liposomes by using the dehydration-rehydration method. Electron microscopic observation demonstrated that the liposomes prepared by this method were large multilamellar vesicles with a diameter of several μm. The encapsulation efficiency was improved by increasing the rehydration temperature to 60°C. The cholesterol/lecithin ratio also affected the efficiency, giving the highest value at a ratio of 1/4 (mol/mol). Some efflux of glucose through the liposomal membranes was observed, particularly for the liposome with a low cholesterol content, but that of IgY was not detected, irrespective of the cholesterol content. Encapsulation reduced the activity loss of the IgY antibodies under acidic conditions. IgY encapsulated in the liposomes was also markedly resistant to pepsin hydrolysis, which usually results in complete loss of activity with unencapsulated IgY, suggesting that liposomal encapsulation is an effective means for protecting IgY under gastric conditions.     

Apolipoprotein B stimulates formation of monocyte-macrophage surface-connected compartments and mediates uptake of low density lipoprotein-derived liposomes into these compartments

Much of the cholesterol that accumulates in atherosclerotic plaques is found within monocyte-macrophages transforming these cells into "foam cells." Native low density lipoprotein (LDL) does not cause foam cell formation. Treatment of LDL with cholesterol esterase converts LDL into cholesterol-rich liposomes having >90% cholesterol in unesterified form. Similar cholesterol-rich liposomes are found in early developing atherosclerotic plaques surrounding foam cells. We now show that cholesterol-rich liposomes produced from cholesterol esterase-treated LDL can cause human monocyte-macrophage foam cell formation inducing a 3-5-fold increase in macrophage cholesterol content of which >60% is esterified. Although cytochalasin D inhibited LDL liposome-induced macrophage cholesteryl ester accumulation, LDL liposomes did not enter macrophages by phagocytosis. Rather, the LDL liposomes induced and entered surface-connected compartments within the macrophages, a unique endocytic pathway in these cells that we call patocytosis. LDL liposome apoB rather than LDL liposome lipid mediated LDL liposome uptake by macrophages. This was shown by the findings that: 1) protease treatment of the LDL liposomes prevented macrophage cholesterol accumulation; 2) liposomes prepared from LDL lipid extracts did not cause macrophage cholesterol accumulation; and 3) purified apoB induced and accumulated within macrophage surface-connected compartments. Although apoB mediated the macrophage uptake of LDL liposomes, this uptake did not occur through LDL, LDL receptor-related protein, or scavenger receptors. Also, LDL liposome uptake was not sensitive to treatment of macrophages with trypsin or heparinase. Cholesterol esterase-mediated transformation of LDL into cholesterol-rich liposomes is an LDL modification that: 1) stimulates uptake of LDL cholesterol by apoB-dependent endocytosis into surface-connected compartments, and 2) causes human monocyte-macrophage foam cell formation.     

Effect of cholesterol on sensitivity of lecithin liposomes to surface-active substances

The effect of cholesterol incorporation into multilamellar egg lecithin liposomes on the liposomes sensitivity toward N-acyl derivatives of amino acids was examined. Free energy of intermolecular interaction between lecithin head groups in the bilayer is estimated as 3.8 ± 0.1 kcal/mol.     

Protein-stimulated exchange of phosphatidylcholine between intact erythrocytes and various membrane systems

Phosphatidylcholine specific exchange protein from beef liver was found to catalyze the exchange of phosphatidylcholine between intact rat and human erythrocytes and various artificial membranes. Both multilamellar liposomes and single bilayer vesicles prepared from egg lecithin, cholesterol and phosphatidic acid (46:50:4, mol/mol) appeared to be effective phospholipid donor systems. Some merits and disadvantages of the various donor systems are discussed.     

Physical and chemical characteristics of apolipoprotein A-I-lipid complexes produced by Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene

Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene linked to the human metallothionein gene promoter region secrete large quantities of apolipoprotein A-I (7.1 +/- 0.4% total secreted protein) in the presence of zinc. Approx. 16% of the secreted apolipoprotein A-I is complexed with lipid and can be isolated ultracentrifugally at d less than or equal to 1.21 g/ml. The latter complexes are composed of discs and vesicles as judged by electron microscopy and can be further separated by column chromatography into three fractions: fraction I, mostly vesicles (60-260 nm) and large discs (18-20 nm diameter); fraction II, discs 14.2 +/- 2.6 nm diameter; and fraction III, nonresolvable by electron microscopy. The latter fraction is extremely lipid-poor (94% protein, 6% phospholipid); in contrast, the protein, phospholipid and unesterified cholesterol content for the other fractions are 43, 33 and 24%, respectively, for fraction I and 53, 33 and 14%, respectively, for fraction II. Fraction II particles contain three and four apolipoprotein A-Is per particle as determined by protein crosslinking while large structures in fraction I contain primarily six to seven apolipoprotein A-Is per particle. Following incubation with purified lecithin: cholesterol acyltransferase, discoidal particles were transformed into apparent spherical particles 12.9 +/- 3.4 nm diameter; this transformation coincided with 19-21% conversion of unesterified cholesterol to esterified cholesterol. The apolipoprotein A-I-lipid complexes isolated from Chinese hamster ovary cell media are similar to nascent HDL found in plasma of lecithin:cholesterol acyltransferase-deficient patients and those secreted by the human hepatoma line, Hep G2. The ability of the Chinese hamster ovary cell nascent HDL-like particles to undergo transformation in the presence of purified lecithin:cholesterol acyltransferase indicates that they are functional particles.     

Permeability properties of liposomes prepared from dipalmitoyllecithin, dimyristoyllecithin, egg lecithin, rat liver lecithin and beef brain sphingomyelin

Liposomes have been prepared from dipalmitoyllecithin, dimyristoyllecithin, egg lecithin, rat liver lecithin and beef brain sphingomyelin.Permeability properties of liposomes thus prepared were studied toward glucose. The glucose permeability of liposomes with saturated lecithins (dipalmitoyllecithin and dimyristoyllecithin) and sphingomyelin appears to be more strongly temperature dependent than that of liposomes with lecithin containing unsaturated fatty acyl chains (egg and rat liver lecithins). The permeability of glucose through vesicles of dipalmitoyllecithin or dimyristoyllecithin was enhanced drastically at their transition temperatures, while the incorporation of about 25 mole% of egg lecithin into liposomes of saturated lecithins suppressed the enhanced permeation rates of glucose above the transition temperatures.The incorporation of small amounts of cholesterol enhanced the temperature-dependent permeability of glucose through the bilayer of saturated lecithins or sphingomyelin. This tendency was best shown in the case of dipalmitoyl-lecithin, in which 20 mole% of cholesterol had the most stimulating effect on the temperature-dependent permeability. The introduction of more than 33 mole% of cholesterol showed, however, reduced effects on the temperature-dependent permeability through liposomes with saturated lecithins or sphingomyelin. It was also shown that cholesterol had a much larger effect on the regulation of the temperature-dependent permeability of liposomes prepared with saturated lecithins or sphingomyelin than on that of liposomes prepared with phospholipids containing unsaturated fatty acids.     

Delivery of liposome membrane-associated sterols through silastic membranes

The transport of sterols incorporated into the lecithin bilayer of small unilamellar liposomes through a model membrane was studied. A two-chamber diffusion cell containing liposomes with incorporated [4-¹⁴C]cholesterol or β-[4-¹⁴C]sitosterol in the donor chamber and liposomes with unlabeled cholesterol in the receiver chamber was used. The permeability coefficients of the sterols through silastic rubber membranes which served as a model membrane were measured. The permeability for cholesterol incorporated into liposomes in a phosphatidyl choline/cholesterol molar ratio of 1 : 1, produced by sonication for 1 h, and subsequent centrifugation at 100000 × g for 1 h, was 1.6 · 10^?8 cm sec^?1. Dilution of the liposome suspension did not change the permeability coefficient significantly. The permeability coefficient of sitosterol incorporated into liposomes was about 4-times smaller than that of cholesterol. These results suggest that the sterols were delivered to the silastic membrane by the intact liposomes and that free solute was not involved in the transport to the membrane to a significant degree. The large differences in the permeability coefficients between cholesterol and sitosterol indicate that an aqueous interfacial barrier was crossed by the sterol during the delivery to the membrane.     

Activation of lecithin: cholesterol acyltransferase by apolipoproteins E-2, E-3, and A-IV isolated from human plasma

Apolipoprotein A-IV, apolipoprotein E-2 and apolipoprotein E-3 were individually incorporated into defined phosphatidylcholine/cholesterol liposomes for study of lecithin:cholesterol acyltransferase activation. Enzyme activities obtained with these liposomes were compared with that from liposomes containing purified apolipoprotein A-I. Apolipoprotein A-IV, apolipoprotein E-2, and apolipoprotein E-3 all activated lecithin:cholesterol acyltransferase. With purified enzyme and with egg yolk phosphatidylcholine as the acyl donor, maximal activation was obtained at a concentration of approximately 0.5 nmol for apolipoprotein A-IV and 0.4 nmol for the apolipoprotein E isoforms. Apolipoprotein A-IV was approximately 25% as efficient as apolipoprotein A-I for the activation of purified enzyme; apolipoprotein E-2 was 40% as efficient, and apolipoprotein E-3, 30%. Similar activation results were obtained using plasma as the enzyme source. Analysis of the plasma of patients with absence of apolipoprotein A-I or with only trace amounts of apolipoprotein A-I exhibited a reduced rate of cholesterol esterification and lecithin:cholesterol acyltransferase activity that was proportional to the reduced level of the enzyme's mass. These results indicate that apolipoprotein A-IV and apolipoprotein E may serve as physiological cofactors for the enzyme reaction.     

Liposomalization of lactoferrin enhanced its anti-tumoral effects on melanoma cells

A number of studies have reported the anti-tumoral activity of lactoferrin, a property mediated by a variety of mechanisms such as inhibitory effects on tumor cell growth, NK cell activation, and enhancement of apoptosis. Liposomes are known to be an efficient drug delivery system which can enhance the therapeutic potential of the encapsulated compounds. We have used positively charged liposomes composed of phosphatidylcholine (PC), dioleoylphosphatidylethanolamine (DOPE), cholesterol (Chol) and stearylamine (SA) (6:1:2:1 M ratio) as a carrier system for bovine iron-free Lf (ApoBLf), and compared the in vitro effect of free and liposome-entrapped ApoBLf on the growth and morphology of murine melanoma B16-F10 cells. Liposomal formulation of ApoBLf was found to enhance the capacity of the protein to inhibit the cell proliferation by affecting cell cycle progression. The effect appeared to be due to the capacity of liposomes to increase the uptake of the protein and its accumulation into cells and probably to protect it from degradation, as revealed by fluorescence microscopy and flow cytometry. Our results demonstrate the ability of liposomes to improve the anti-tumor activity of Lf and suggest that liposomal protein may have a potential therapeutic use in the prevention and/or treatment of cancer diseases.     

Changes of membrane permeability due to extensive cholesterol depletion in mammalian erythrocytes

55% of the total membrane cholesterol could be removed from porcine, bovine and human erythrocytes by incubating the cells in suspensions of lecithin liposomes. Up to 30% depletion, membrane permeability remained unaltered; more extensive depletion induced a marked increase of the transfer rates of nonelectrolytes and of organic acids penetrating by nonionic diffusion. This biphasic response of permeability to cholesterol depletion, which has not been observed in artificial lipid membranes, may be related to the heterogeneity of the erythrocyte membrane lipids or to a pool of cholesterol not interacting with the phospholipids.     

Plasmodium falciparum: one-step growth in a semi-defined medium and the stimulatory effect of human seric lipoproteins and liposomes

The ring stages of Plasmodium falciparum within red blood cells cultured with complete medium stop growing when transferred to a basic medium containing RPMI plus fatty acid-free bovine serum albumin and dialyzable factors from human serum. Growth and multiplication can be partially restored by the addition of lipoprotein fractions prepared from human serum. No specificity was observed with subclasses of lipoproteins. Synthetic liposomes containing lecithin, oleic acid, and cholesterol mimic the effect of lipoproteins.     

Cytotoxic effect of unilamellar detergent dialysis liposomes on Trypanosoma brucei and Trypanosoma congolense bloodstream forms in vitro

Liposomes from egg yolk lecithin and egg yolk lecithin/ganglioside are cytotoxic for Trypanosoma brucei and Trypanosoma congolense bloodstream forms in vitro. The trypanocidal effect is influenced by the liposome age and concentration. This effect is diminished in the presence of whole blood in vitro and could not be observed in vivo. Freeze-fractured parasite membrane showed an intramembranous particle aggregation after incubation with liposomes. Liposomes from egg yolk lecithin kill trypanosomes more rapidly than do liposomes from egg yolk lecithin/cholesterol.     

Down-regulation of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase activity with highly purified liposomal cholesterol.

Chinese hamster ovary-215 cells (CHO-215) cannot synthesize C27 and C28 sterols because of a defect in the reaction that decarboxylates 4-carboxysterols [Plemenitas, A., Havel, C.M. & Watson, J.A. (1990) J. Biol. Chem. 265, 17012-17017]. Thus, CHO-215 cell growth is dependent on an exogenous metabolically functional source of cholesterol. We used CHO-215 cells to (a) determine whether highly purified (> 99.5%) cholesterol, in egg lecithin liposomes, could down-regulate derepressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and if so (b) determine whether the loss in reductase catalytic activity correlated kinetically with the synthesis and accumulation of detectable oxycholesterol derivatives. Liposomal cholesterol (26-39 microM) supported maximum CHO-215 growth and initiated suppression of HMG-CoA reductase activity at concentrations greater than 50 microM. Maximum suppression (50-60%) of reductase activity was achieved with 181.3 microM liposomal cholesterol in 6 h. Also, regulatory concentrations of highly purified liposomal [3H]cholesterol were not converted (biologically or chemically) to detectable levels of oxy[3H]cholesterol derivatives during 3-6 h incubations. Lastly, a broad-spectrum cytochrome P450 inhibitor (miconazole) had no effect on liposomal cholesterol-mediated suppression of HMG-CoA reductase activity. These observations established that (a) highly purified cholesterol, incorporated into egg lecithin liposomes, can signal the down-regulation of derepressed mammalian cell HMG-CoA reductase activity and (b) if oxycholesterol synthesis was required for liposomal cholesterol-mediated down-regulation, the products had to be more potent than 24-, 25-, or 26-/27-hydroxycholesterol.     

Comparison of cholesterol-phospholipid interaction in bilayers of liposomes and the red cell membrane

The energetics of interactions of cholesterol with phospholipid in simple liposome bilayers were compared with those in the bilayer of the human erythrocyte membrane, by measuring cholesterol distribution between erythrocytes and liposomes prepared from their whole phospholipid extract. With liposomes of a range of initial cholesterol contents, the equilibrium value for $\text{r}$, the ratio of cholesterol/phospholipid in the liposomes to that in the cells, is in the range 1.1–1.2. The closeness of this value to 1.0 indicates that overall cholesterol-phospholipid interaction in the cell membrane is similar to that in liposomes. However, while the deviation from 1.0 is small, and could arise from average cholesterol-phospholipid interactions in the membrane being only 0.06 to 0.1 kcal · mol^?1 weaker than in liposomes, it could also result from 10 to 20% of the cell membrane phospholipid being unavailable to mix with cholesterol.     

Characterization of high-density lipoprotein binding and cholesterol efflux in cultured mouse adipose cells

Binding of high-density lipoproteins to cultured mouse Ob1771 adipose cells was studied, using labeled human HDL3, mouse HDL and apolipoprotein AI- or AII-containing liposomes. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 18, 42, 30 and 3.4 micrograms/ml, whereas the maximal binding capacities were found to be 160, 100, 90 and 21 ng/mg of cell protein. Apoprotein AI not inserted into liposomes did not bind. The binding of 125I-HDL3 was competitively inhibited by apolipoprotein AI-containing liposomes greater than mouse HDL greater than HDL3. The binding of 125I-labeled apolipoprotein AI- and 125I-labeled apolipoprotein AII-containing liposomes was competitively inhibited by HDL3, apolipoprotein AI- and apolipoprotein AII-containing liposomes. Dimyristoylphosphatidylcholine liposomes containing or not cholesterol did not interfere with the binding of labeled HDL3 or apolipoprotein-containing liposomes. Binding studies on crude membranes of Ob1771 adipose cells revealed the presence of intracellular binding sites for LDL and HDL3. Thus, adipose cells have specific binding sites for apolipoprotein E-free HDL and apolipoprotein AI (or AII) is the ligand for these binding sites. Long-term exposure of adipose cells to LDL cholesterol as a function of LDL concentration led to an accumulation of cellular unesterified cholesterol. This process was saturable and reversible as a function of time and concentration by exposure to HDL3 or apolipoprotein AI-containing liposomes, whereas apolipoprotein AII-containing liposomes did not promote any cholesterol efflux. Since long-term exposure of adipose cells to LDL and HDL3 did not affect the number of apolipoprotein B,E receptors and apolipoprotein E-free binding sites, respectively, it appears that adipose cells do not show efficient cholesterol homeostasis and thus could accumulate or mobilize unesterified cholesterol.     

Self-adaptive modification of red-cell membrane lipids in lecithin: Cholesterol acyltransferase deficiency. Lipid analysis and spin labeling

In a patient with lecithin: cholesterol acyltransferase deficiency, free cholesterol was markedly increased, and esterified cholesterol was diminished. In the patient's plasma, an increase in phosphatidylcholine (PC) and a decrease in sphingomyelin were observed. Concomitantly, an increase in a shorter acyl chain 16:0 was noted in PC, sphingomyelin and phosphatidylethanolamine (PE). In contrast to these results, longer chains such as 22:0 and 24:0 were decreased, especially in sphingomyelin. Unsaturated double bonds such as 18:1 was also increased in PC and PE. In the red-cell membrane lipids, the increase in free cholesterol was counteracted by an increase in PC and by a decrease in sphingomyelin and PE, reflecting changes in the patient's plasma lipids. Increased 16:0 (in PC) and decreased 18:0 and 24:0 were observed. The increased plasma free cholesterol due to metabolic defect (lecithin:cholesterol acyltransferase deficiency) led to decreased red-cell membrane fluidity. This effect appeared to be counteracted by changing phospholipid composition (increased PC and decreased sphingomyelin and PE), by increasing shorter chains (16:0), by decreasing longer chains (18:0 and 24:0) and by increasing unsaturated double bonds (18:2). These results can be interpreted as a self-adaptive modification of lecithin:cholesterol acyltransferase deficiency-induced red-cell membrane abnormalities, to maintain normal membrane fluidity. This speculation was supported by the ESR spin-label studies on the patient's membrane lipids. The normal order parameters in intact red cells and in total lipid liposomes were decreased if cholesterol-depleted membrane liposomes were prepared. Thus, the hardening effect of cholesterol appeared to be counteracted by the softening effects described above. Overall membrane fluidity in intact red cells of the lecithin:cholesterol acyltransferase-deficient patient was maintained normally, judged by order parameters in ESR spin-label studies.