The influence of cholesterol and charge on the membrane domains of alkylphospholipid liposomes as studied by EPR

Alkylphospholipids are physiologically active derivatives of lipids effective in the treatment of breast cancer. Among them, octadecyl-(1,1-dimethyl-4-piperidino-4-yl)-phosphate (OPP) was demonstrated recently to have the strongest antitumor effect in micellar as well as in sterically stabilised liposome suspension with a low cholesterol content. In this work electron paramagnetic resonance (EPR) was used to study the influence of cholesterol, charge, and sterical stabilisation by PEG2000DSPE on the domain structure and fluidity characteristics of the membrane of OPP liposomes. As a spin probe 5-doxylpalmitoyl methyl ester was used. By computer simulation of the EPR spectra it was found that the experimental spectra are composed of three spectral components, which were attributed to three types of domains with different fluidity characteristics. The EPR parameters as well as the proportions of the individual domains were found to be mainly dependent on the amount of cholesterol, and only to a minor degree on charge and sterical stabilisation. There was a pronounced increase in the proportion of membrane domains with low order parameter, when the molar ratio of cholesterol to OPP was decreased below 1. At the same time the order parameters of all domains decreased, pointing to a transition from a less to a more fluid membrane organisation. These results coincide with an improved therapeutic activity of formulations with a low molar ratio of cholesterol to OPP and indicates that the fluidity characteristics of the membrane may be important for the effectiveness of liposomal alkylphospholipids against breast cancer cells.     

The synaptophysin/synaptobrevin interaction critically depends on the cholesterol content

Synaptophysin interacts with synaptobrevin in membranes of adult small synaptic vesicles. The synaptophysin/synaptobrevin complex promotes synaptobrevin to built up functional SNARE complexes thereby modulating synaptic efficiency. Synaptophysin in addition is a cholesterol-binding protein. Depleting the membranous cholesterol content by filipin or beta-methylcyclodextrin (beta-MCD) decreased the solubility of synaptophysin in Triton X-100 with less effects on synaptobrevin. In small synaptic vesicles from rat brain the synaptophysin/synaptobrevin complex was diminished upon beta-MCD treatment as revealed by chemical cross-linking. Mice with a genetic mutation in the Niemann-Pick C1 gene developing a defect in cholesterol sorting showed significantly reduced amounts of the synaptophysin/synaptobrevin complex compared to their homo- or heterozygous littermates. Finally when using primary cultures of mouse hippocampus the synaptophysin/synaptobrevin complex was down-regulated after depleting the endogenous cholesterol content by the HMG-CoA-reductase inhibitor lovastatin. Alternatively, treatment with cholesterol up-regulated the synaptophysin/synaptobrevin interaction in these cultures. These data indicate that the synaptophysin/synaptobrevin interaction critically depends on a high cholesterol content in the membrane of synaptic vesicles. Variations in the availability of cholesterol may promote or impair synaptic efficiency by interfering with this complex.     

Interaction of solid liposomes with epithelial cells

Binding of fluorescein-containing solid liposomes to cultured epithelial cells was studied. It was found that liposomes were preferentially attached to the contact-free lateral edges of epithelial cells but not to the upper surfaces of these cells and not to the edges forming lateral cell-cell contacts. This preferential binding to cell margins was characteristic only of the solid liposomes but not of the fluid ones. Binding of solid liposomes to the cells could be prevented and reversed by trypsin. Possible mechanisms of special adhesive properties of the surface of contact-free cell edges are discussed.     

Interaction of liposomes with Zymomonas mobilis cells

Abstract Zymomonas mobilis (strain ZM4), an ethanoltolerant bacterium, interacts efficiently with liposomes consisting of Escherichia coli phospholipids. The parameters of the cell-liposome interaction have been determined. The process depends on temperature, presence of divalent cation, pH, liposome/cell ratio and is insensitive to pre-treatment of ZM4 cells with EDTA. Optimal transfer of exogenous phospholipids into cells was achieved at 37°C and pH 6.6, when the interaction medium contained 20 mM CaCl₂.     

Interaction of liposomes with Kupffer cells in vitro

We investigated the interaction of liposomes with rat Kupffer cells in monolayer maintenance culture. The liposomes (large unilamellar vesicles, LUV) were composed of 14C-labelled phosphatidylcholine, cholesterol and phosphatidylserine (molar ratio 4:5:1) and contained either 3H-labelled inulin or 125I-labelled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. After 2-3 days in culture the cells exhibited optimal uptake capacity. The uptake process showed saturation kinetics, maximal uptake values amounting to 2 nmol of total liposomal lipid/h/10(6) cells. This is equivalent to 1500 vesicles per cell. The presence of fetal calf serum (FCS) during incubation increased uptake nearly two-fold, whereas freshly isolated rat serum had no effect. The binding of the liposomes to the cells caused partial release of liposomal contents (about 15-20%) both at 4 degrees C and at 37 degrees C. In the presence of metabolic inhibitors the uptake at 37 degrees C was reduced to about 20% of the control values. Inulin and lipid label became cell-associated at similar rates and extents, whereas the association of albumin label gradually decreased after attaining a maximum at relatively low values. When, after 1 h incubation, the liposomes were removed continued incubation for another 2 h in absence of liposomes led to an approx. 30% release of cell-associated lipid label into the medium in water-soluble form. Under identical conditions as much as 90% of the cell-associated albumin label was released in acid-soluble form. Contrarily, the inulin label remained firmly cell-associated under these conditions. From these results we conclude that Kupffer cells in monolayer culture take up liposomes primarily by way of an adsorptive endocytic mechanism. This conclusion was confirmed by morphological observations on cells incubated with liposomes containing fluorescein isothiocyanate (FITC) dextran or horseradish peroxidase as markers for fluorescence microscopy and electron microscopy, respectively.     

Yeast virus propagation depends critically on free 60S ribosomal subunit concentration.

Over 30 MAK (maintenance of killer) genes are necessary for propagation of the killer toxin-encoding M1 satellite double-stranded RNA of the L-A virus. Sequence analysis revealed that MAK7 is RPL4A, one of the two genes encoding ribosomal protein L4 of the 60S subunit. We further found that mutants with mutations in 18 MAK genes (including mak1 [top1], mak7 [rpl4A], mak8 [rpl3], mak11, and mak16) had decreased free 60S subunits. Mutants with another three mak mutations had half-mer polysomes, indicative of poor association of 60S and 40S subunits. The rest of the mak mutants, including the mak3 (N-acetyltransferase) mutant, showed a normal profile. The free 60S subunits, L-A copy number, and the amount of L-A coat protein in the mak1, mak7, mak11, and mak16 mutants were raised to the normal level by the respective normal single-copy gene. Our data suggest that most mak mutations affect M1 propagation by their effects on the supply of proteins from the L-A virus and that the translation of the non-poly(A) L-A mRNA depends critically on the amount of free 60S ribosomal subunits, probably because 60S association with the 40S subunit waiting at the initiator AUG is facilitated by the 3' poly(A).     

Interaction of cytoplasmic L-glycerol-3-phosphate dehydrogenase with liposomes

NAD-linked L-glycerol-3-phosphate dehydrogenase binds to phosphatidylcholine liposomes as shown by the changes in the properties of both the enzyme and the membrane. The surface potential and the fluidity of the liposome membrane (monitored at the 5th C atom depth) change due to the presence of the enzyme, whereas the enzyme is activated by the liposomes. These findings suggest the occurrence of peripheral protein-lipid interactions.     

Effectiveness of incorporation of spermine-condensed viral DNA into liposomes. Interaction of liposomes with cells

DNA from bacteriophage lambda or monkey adenovirus type 7 have been condensed with spermine and included into three types of liposomes: ethereal, obtained by inverted phases technique and by Ca++ fusion. Compact DNA form presents a tor with 100 nm external diameter and 430 nm width. It can be included into 100 nm or larger liposomes. Total preparation contains 15-18% of the required liposomes. Liposomal fractions with included DNA were separated from empty liposomes by step gradient of ficoll 400. Liposomal fraction having included DNA contains 15-18% of common lipid. Liposomal interaction with monkey cell cultures has been studied.     

Interaction of cells from murine organs with liposomes of various composition

The distribution of liposomes prepared from total mouse liver lipids and containing (3H)-labelled platelet activation factor in mouse organs was studied. It was shown that the majority of intraperitoneally injected liposomes prepared from total mouse liver lipids were transported to mouse liver and spleen. The interaction of liposomes with spleen cells in vitro revealed that the affinity of liposomes prepared from total spleen macrophage or total spleen lymphocyte lipids for mouse spleen cells was much higher than that of liposomes prepared from a model lipid mixture. The liposome binding to isolated spleen macrophages or lymphocytes was much higher than the liposome uptake by these cells in the total population of mouse spleen cells.     

Interaction of liposomes bearing a lipophilic doxorubicin prodrug with tumor cells

When used as nanosized carriers, liposomes enable targeted delivery and decrease systemic toxicity of antitumor agents significantly. However, slow unloading of liposomes inside cells diminishes the treatment efficiency. The problem could be overcome by the adoption of lipophilic prodrugs tailored for incorporation into lipid bilayer of liposomes. We prepared liposomes of egg yolk phosphatidylcholine and yeast phosphatidylinositol bearing a diglyceride conjugate of an antitumor antibiotic doxorubicin (a lipophilic prodrug, DOX-DG) in the membrane to study how these formulations interact with tumor cells. We also prepared liposomes of rigid bilayer-forming lipids, such as a mixture of dipalmitoylphosphatidylcholine and cholesterol, bearing DOX in the inner water volume, both pegylated (with polyethylene glycol (PEG) chains exposed to water phase) and non-pegylated. Efficiency of binding of free and liposomal doxorubicin with tumor cells was evaluated in vitro using spectrofluorimetry of cell extracts and flow cytometry. Intracellular traffic of the formulations was investigated by confocal microscopy; co-localization of DOX fluorescence with organelle trackers was estimated. All liposomal formulations of DOX were shown to distribute to organelles retarding its transport to nucleus. Intracellular distribution of liposomal DOX depended on liposome structure and pegylation. We conclude that the most probable mechanism of the lipophilic prodrug penetration into a cell is liposome-mediated endosomal pathway.     

Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

Background

Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis.

Methods

Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age.

Results

Membrane cholesterol measurements are elevated in both R117H and ΔF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTR_inh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility.

Conclusions

The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.     

Interaction of albumin and phospholipid:cholesterol liposomes in growth of Mycoplasma spp

Mycoplasma spp., sterol and fatty acid auxotrophs, are conventionally grown in complex media containing high concentrations of serum. Serum supplies the required lipids, but its presence complicates studies on the metabolism and antigenicity of mycoplasmas as well as the membrane dynamics of these organisms. In the present work, fetal bovine serum was replaced with dilipidated albumin and liposomes containing high concentrations of cholesterol. The liposomes were produced from phosphatidylcholine which contained other lipid species, including phosphatidylethanolamine, phosphatidylglycerol, and cholesterol. Other liposomes containing cholesterol and one phospholipid yielded significantly less growth of Mycoplasma gallisepticum, indicating that several phospholipids are required to achieve growth levels comparable to those obtained with complex medium. The sources and concentrations of cholesterol, albumin, phosphatidylcholine, and other phospholipids and the interactions among them were important affectors of mycoplasmal growth. Optimal lipid and albumin conditions established for M. gallisepticum were then used to propagate five diverse Mycoplasma spp. to growth levels which equalled or surpassed those obtained with medium containing 17% fetal bovine serum.     

Syntaxin and VAMP association with lipid rafts depends on cholesterol depletion in capacitating sperm cells

Sperm cells represent a special exocytotic system since mature sperm cells contain only one large secretory vesicle, the acrosome, which fuses with the overlying plasma membrane during the fertilization process. Acrosomal exocytosis is believed to be regulated by activation of SNARE proteins. In this paper, we identified specific members of the SNARE protein family, i.e., the t-SNAREs syntaxin1 and 2, and the v-SNARE VAMP, present in boar sperm cells. Both syntaxins were predominantly found in the plasma membrane whereas v-SNAREs are mainly located in the outer acrosomal membrane of these cells. Under non-capacitating conditions both syntaxins and VAMP are scattered in well-defined punctate structures over the entire sperm head. Bicarbonate-induced in vitro activation in the presence of BSA causes a relocalization of these SNAREs to a more homogeneous distribution restricted to the apical ridge area of the sperm head, exactly matching the site of sperm zona binding and subsequent induced acrosomal exocytosis. This redistribution of syntaxin and VAMP depends on cholesterol depletion and closely resembles the previously reported redistribution of lipid raft marker proteins. Detergent-resistant membrane isolation and subsequent analysis shows that a significant proportion of syntaxin emerges in the detergent-resistant membrane (raft) fraction under such conditions, which is not the case under those conditions where cholesterol depletion is blocked. The v-SNARE VAMP displays a similar cholesterol depletion-dependent lateral and raft redistribution. Taken together, our results indicate that redistribution of syntaxin and VAMP during capacitation depends on association of these SNAREs with lipid rafts and that such a SNARE-raft association may be essential for spatial control of exocytosis and/or regulation of SNARE functioning.     

Interaction of ferricytochrome c with liposomes

The binding of ferricytochrome c to liposomes consisting of phosphatidylcholine mixtures with cardiolipin (3:1) or phosphatidylserine (3:1) has been investigated. Experimental data have been analyzed in terms of two-dimensional models of large ligand adsorption. The equilibrium parameters of ferricytochrome c interaction with a phospholipid bilayer are determined.     

Interaction of isopropylthioxanthone with phospholipid liposomes

Isopropylthioxanthone (ITX) is a highly lipophilic molecule which can be released in foods and beverages from the packages, where it is present as photoinitiator of inks in printing processes. Recently it was found in babies milk, and its toxicity cannot be excluded. The structure of the molecule suggests a possible strong interaction with the lipid moiety of biological membranes, and this is the first study of its effects on phospholipid organization, using differential scanning calorimetry (DSC) and spin labelling techniques. The data obtained with multilamellar liposomes of saturated phospholipids of different length, with and without cholesterol, point out that the molecule changes the lipid structure; in particular, in the gel state, behaving like a disordering agent it increases the mobility of the bilayer, while, in the fluid state, tends to rigidify the membrane, in a cholesterol like way. This behavior supports the hypothesis that ITX experiences a relocation process when the lipid matrix passes from the gel to the fluid state.     

Interaction of fluoxetine with phosphatidylcholine liposomes

Fluoxetine (Prozac) is one of the latest of a new generation of antidepressants, approved by FDA in 2002. The interactions of fluoxetine with multilamellar liposomes of pure phosphatidylcholine (PC) or containing cholesterol 10% molar were studied as a function of the lipid chain lengths, using differential scanning calorimetry and spin labelling EPR techniques. The DSC profiles of the gel-to-fluid state transition of liposomes of DMPC (C14:0) are broadened and shifted towards lower temperatures at increasing dopant concentrations and, with less than 10% fluoxetine, any detectable transition is destroyed. The broadened profiles and the lowered transition temperatures demonstrate that both the size and the packing of the cooperative units undergoing the transition are modified by fluoxetine, leading to a looser and more flexible bilayer. No phase separation was observed. The effects of fluoxetine on the thermotropic phase behaviour of DPPC (C16:0) and, even more, of DSPC (C18:0) are different from that of DMPC. In fact, in the former cases, two peaks appeared at increasing dopant concentrations, suggesting the occurrence of a phase separation phenomenon, which is a sign of a binding of fluoxetine in the phosphate region. In cholesterol containing membranes, fluoxetine, even at low concentrations, leads to a general corruption of the membrane, both in terms of packing and cooperativity, and the formation of any new phase is no longer observable. EPR spectra reflect the disordered motion of acyl chains in the bilayer. It was found that fluoxetine lowers the order of the lipid chains mainly in correspondence of the fifth carbon position of SASL, indicating a possible accumulation near the interfacial region.     

Interaction of cationic liposomes and their DNA complexes with monocytic leukemia cells

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. We examined the relationship between the characteristics of the lipoplexes, their mode of interaction with monocytic THP-1 cells and their ability to transfect these cells. We determined the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and its mixtures with neutral lipids), and lipoplexes at different (+/-) charge ratios. As the (+/-) charge ratio of the lipoplexes decreased to (1/1), a significant reduction in zeta potential and an increase in size was observed. The increase in size resulted from fusion between liposomes promoted by DNA, as demonstrated by a lipid mixing assay, and from aggregation of the complexes. Interaction of liposomes and lipoplexes with THP-1 cells was assessed by monitoring lipid mixing ('fusion') as well as binding and cell association. While no lipid mixing was observed with the 1/2 (+/-) lipid/DNA complexes, lipoplexes with higher (+/-) charge ratios underwent significant fusion in conjunction with extensive cell binding. Liposome binding to cells was dependent on the positive charge of the liposomes, and their fusion could be modulated by the co-lipid. DOTAP/phosphatidylethanolamine (1:1) liposomes fused with THP-1 cells, unlike DOTAP/phosphatidylcholine (1:1) liposomes, although both liposome types bound to the cells to a similar extent. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. The presence of serum increased the size of the cationic liposomes, but not that of the lipoplexes. Low concentrations of serum (3%) completely inhibited the fusion of cationic liposomes with cells, while inhibiting binding by only 20%. Our results suggest that binding of cationic liposomes and lipoplexes to cells is governed primarily by electrostatic interactions, whereas their fusion is regulated by the lipid composition and sterically favorable interactions with cell surface molecules. In addition our results indicate no correlation between fusion of the lipoplexes with the plasma membrane and the levels of transfection.     

Interaction of antiinflammatory drugs with EPC liposomes: calorimetric study in a broad concentration range

Isothermal titration calorimetry was used to characterize and quantify the partition of indomethacin and acemetacin between the bulk aqueous phase and the membrane of egg phosphatidylcholine vesicles. Significant electrostatic effects were observed due to binding of the charged drugs to the membrane, which implied the use of the Gouy-Chapman theory to calculate the interfacial concentrations. The binding/partition phenomenon was quantified in terms of the partition coefficient (K(p)), and/or the equilibrium constant (K(b)). Mathematical expressions were developed, either to encompass the electrostatic effects in the partition model, or to numerically relate partition coefficients and binding constants. Calorimetric titrations conducted under a lipid/drug ratio >100:1 lead to a constant heat release and were used to directly calculate the enthalpy of the process, DeltaH, and indirectly, DeltaG and DeltaS. As the lipid/drug ratio decreased, the constancy of reaction enthalpy was tested in the fitting process. Under low lipid/drug ratio conditions simple partition was no longer valid and the interaction phenomenon was interpreted in terms of binding isotherms. A mathematical expression was deduced for quantification of the binding constants and the number of lipid molecules associated with one drug molecule. The broad range of concentrations used stressed the biphasic nature of the interaction under study. As the lipid/drug ratio was varied, the results showed that the interaction of both drugs does not present a unique behavior in all studied regimes: the extent of the interaction, as well as the binding stoichiometry, is affected by the lipid/drug ratio. The change in these parameters reflects the biphasic behavior of the interaction-possibly the consequence of a modification of the membrane's physical properties as it becomes saturated with the drug.     

Dendritic cell interaction with Candida albicans critically depends on N-linked mannan

The fungus Candida albicans is the most common cause of mycotic infections in immunocompromised hosts. Little is known about the initial interactions between Candida and immune cell receptors, because a detailed characterization at the structural level is lacking. Antigen-presenting dendritic cells (DCs), strategically located at mucosal surfaces and in the skin, may play an important role in anti-Candida protective immunity. However, the contribution of the various Candida-associated molecular patterns and their counter-receptors to DC function remains unknown. Here, we demonstrate that two C-type lectins, DC-SIGN and the macrophage mannose receptor, specifically mediate C. albicans binding and internalization by human DCs. Moreover, by combining a range of C. albicans glycosylation mutants with receptor-specific blocking and cytokine production assays, we determined that N-linked mannan but not O-linked or phosphomannan is the fungal carbohydrate structure specifically recognized by both C-type lectins on human DCs and directly influences the production of the proinflammatory cytokine IL-6. Better insight in the carbohydrate recognition profile of C-type lectins will ultimately provide relevant information for the development of new drugs targeting specific fungal cell wall antigens.