An EPR spin probe study of liposomes from sunflower and soybean phospholipids

Comparative properties of lecithin-based liposomes prepared from the mixed phospholipids of sunflower seeds, soybean and egg yolk were investigated by electron paramagnetic resonance (EPR) spectroscopy. For these investigations, stable nitroxide radicals, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 5,7-dimethyladamantane-1-carboxylate (DMAC-TEMPO), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) were used as spin probes. Binding of the spin probes to the liposome membranes resulted in a substantial increase of the apparent rotational diffusion correlation times. The EPR spectra of the incorporated nitroxides underwent temperature-dependent changes. For every spin probe, values of apparent enthalpy and entropy of activation were calculated from the temperature dependence of rotational diffusion correlation times via Arrhenius equation. In case of DMAC-TEMPO, the data point to differences between the phospholipid bilayer of liposomes derived from sunflower and soy lecithin, and some similarity between the sunflower and egg yolk liposomes. Anisotropic hyperfine interaction constants of DMAC-TEMPO and 16-DSA included in the liposomes have been analyzed and attributed to different micropolarity of the surroundings of the spin probes. The kinetics of EPR signal decay of DMAC-TEMPO in the presence of 2,2′-azobis(2-amidinopropane) suggest the better stability of the sunflower liposomes to lipid peroxidation as compared to the liposomes prepared from soy lecithin.     

Fatty-acid spin probe interactions with erythrocyte ghosts and liposomes prepared from erythrocyte ghosts

Summary A model for the binding of 5-nitroxide stearate, I(12,3). to human erythrocyte ghosts was developed by comparing spin probe interactions with ghosts and liposomes prepared from ghosts. At low probe/lipid (P/L<1/2500), I(12,3) binds to a similar class of high-affinity, noninteracting sites in both ghosts and liposomes, indicating that lipid moieties are responsible for probe uptake. Saturation occurs in both systems with increasing P/L, and, at higher loading (e.g., P/L=1/360 for ghosts and liposomes), the probe inserts itself at initially dilute sites to form a class of low-affinity sites consisting of clusters of variable size. At still higher P/L ranges (>1/100), much increased probe uptake was observed in ghosts than in liposomes, which was attributed to another class of low-affinity sites, representing nonspecific interactions of I(12,3) with membrane proteins. The nature of the spectral components and ultrafiltration experiments with ghosts labeled at high P/L indicate that both dilute and clustered I(12,3) are due to membrane-incorporated probe.     

Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane‐adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel‐fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large‐scale lipid phase separation and protein segregation in intact, protein‐crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.     

A fluorescence approach of the gamma radiation effects on gramicidin A inserted in liposomes.

The fluorescence of tryptophan residues of gramicidin A (gA), bound to phosphatidylcholine liposomes contains valuable information about local changes in the environment of the molecule induced by gamma radiation. With this work, we aim to demonstrate that the gamma radiation effect on the peptide involves the action of free radicals, derived from water radiolysis and the process of lipid peroxidation. Basically, the methodology consists of the analysis of UV and fluorescence emission spectra of the peptide liposome complexes under control conditions and upon gamma irradiation. Free radical production was impaired by the removal of molecular oxygen or the presence of ethanol in the liposome suspension. The intensity of the tryptophan fluorescence was recorded as a function of the gamma radiation dose in the range of 0-250 Gy and the data were fitted with a single decay exponential function containing an additional constant term (named residual fluorescence). The correlation between the decrease in tryptophan fluorescence emission (D(c) = 80 +/- 10 Gy) and increase in gamma radiation dose indicates the partial damage of the tryptophan side chains of gA. O(2) removal or ethanol addition partially reduced the decay of the tryptophan fluorescence emission involving an indirect action of gamma radiation via a water radiolysis mechanism. The residual fluorescence emission (A(0)) increases in O(2)-free buffer (98 +/- 13) and in 10% ethanol-containing buffer (74 +/- 34) compared to control conditions (23 +/- 5). Varying the dose rate between 1-10 Gy/min at a constant dose of 50 Gy, an inverse dose-rate effect was observed. Thus, our study provides evidence for the lipid peroxidation effect on the tryptophan fluorescence. In conclusion, this article sustains the hypothesis of the connection between the lipid peroxidation and structural changes of membrane proteins induced by gamma radiation. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Higly fusogenic cationic liposomes transiently permeabilize the plasma membrane of HeLa cells

Cationic liposomes can efficiently carry nucleic acids into mammalian cells. This property is tightly connected with their ability to fuse with negatively charged natural membranes (i.e. the plasma membrane and endosomal membrane). We used FRET to monitor and compare the efficiency of lipid mixing of two liposomal preparations — one of short-chained diC14-amidine and one of long-chained unsaturated DOTAP — with the plasma membrane of HeLa cells. The diC14-amidine liposomes displayed a much higher susceptibility to lipid mixing with the target membranes. They disrupted the membrane integrity of the HeLa cells, as detected using the propidium iodide permeabilization test. Morphological changes were transient and essentially did not affect the viability of the HeLa cells. The diC14-amidine liposomes were much more effective at either inducing lipid mixing or facilitating transfection.     

Successful transfection of hepatoma cells after encapsulation of plasmid DNA into negatively charged liposomes

The application of conventional cationic liposomes/DNA complexes in gene transfer was hampered due to their large size, instability, and limited transfection site in vivo. In this report, we described a dialysis-based method and produced small, stable, and negatively charged DNA-containing liposomes composed of low content of cationic lipid and high content of fusogenic lipid. The liposomes were relatively spherical with a condensed core inside, and exhibited small size with narrow particle size distribution. The encapsulation efficiency of the liposomes was 42.53 +/- 2.29%. They were stable and showed enough protective ability to plasmid DNA from degradation after incubation with different amounts of DNase. Twenty-fold higher transfection efficiency for the liposomes was achieved when compared with that of naked plasmid DNA and no toxicities to hepatocellular carcinoma cells were observed. Our results indicate that the negatively charged DNA-containing liposomes can facilitate gene transfer in cultured cells, and may alleviate the drawbacks of the conventional cationic liposomes/DNA complexes for gene delivery in vivo.     

Stability study of stavudine-loaded O-palmitoyl-anchored carbohydrate-coated liposomes

The purpose of this study was to evaluate the physicochemical stability of carbohydrate-anchored liposomes. In the present study, carbohydrate (galactose, fucose, and mannose) was palmitoylated and anchored on the surface of positively charged liposomes (PL). The stabilities of plain neutral liposomes (NL), PL, and O-palmitoyl carbohydrate-anchored liposomes were determined. The effects of storage conditions (4°C±2°C, 25°C±2°C/60%±5% relative humidity [RH], or 40°C±2°C/75%±5% RH for a period of 10, 20, and 30 days) were observed on the vesicle size, shape, zeta potential, drug content, and in vitro ligand agglutination assay by keeping the liposomal formulations in sealed ambercolored vials (10-mL capacity) after flushing with nitrogen. The stability of liposomal formulations was found to be temperature dependent. All the liposomal formulations were found to be stable at 4°C±2°C up to 1 month. Storage at 25°C±2°C/60%±5% RH and 40°C±2°C/75%±5% RH adversely affected uncoated liposomal formulations. Carbohydrate coating of the liposomes could enhance the stability of liposomes at 25°C±2°C/60%±5% RH and 40°C±2°C/75%±5% RH. Published: May 18, 2007     

The permeability of reconstituted liposomes containing the purified lens fiber cell integral membrane proteins MP20, MP26 and MP70

Summary A number of lens fiber cell integral membrane proteins have been localized to junctional regions where they have been proposed to play a role in either mediating or controlling cell-to-cell communication. We have examined the effect of three lens fiber cell membrane proteins, MP20, MP26 and MP70, on the permeability properties of unilamellar phospholipid liposomes. This approach has been previously used to examine the channel-forming properties of MP26. Liposome permeability was determined by measuring the effect of Co²⁺ on the quenching of the fluorescence of N-4-nitrobenzo-2-oxa-1,3 diazole phosphatidyl ethanolamine (NBD-PE)-containing liposomes as described previously by Scaglione and Rintoul (Invest. Ophthalmol. Vis. Sci. 30:961–966, 1989). The effect of all three proteins on liposome permeability was similar. Permeability was dependent on the protein/phospholipid ratio and was not significantly affected by agents known to modify gap junctional permeability in vivo. Glycophorin A, a non-channel-forming integral membrane protein derived from erythrocytes, was also shown to increase the permeability of unilamellar phospholipid liposomes. The ability of a non-channel membrane protein to increase Co²⁺ quenching of NBD-PE-containing liposomes (presumably in a nonspecific manner) indicates that reports describing the permeability of lens membrane protein-containing liposomes should be interpreted with caution in terms of their relationship to cell-to-cell communication.We would like to thank Dr. Rita Meyer for technical assistance with the freeze-fracture electron microscopy, Drs. Wolfgang Baumann and Barbara Malewicz for the purification of bovine lens lipids, and Dr. Gary Nelsestuen for the use of both the fluorescence and photon correlation spectrophotometers as well as for many helpful discussions. This research was supported by NIH grant EY 05684.     

Betacyanins as phenol antioxidants. Chemistry and mechanistic aspects of the lipoperoxyl radical-scavenging activity in solution and liposomes

Reaction kinetics of betanin and its aglycone betanidin towards peroxyl radicals generated from the azo-initiated oxidation of methyl linoleate in methanol and of a heterogeneous aqueous/soybean phosphatidylcholine liposomal system were studied by monitoring formation of linoleic acid hydroperoxides and consumption of the pigments. Betanin was a weak retarder in methanol and an effective chain breaking antioxidant in the liposomal model, indicating that kinetic solvent effects and partition in lipid bilayers may affect its activity. Betanidin behaved as a chain terminating antioxidant in both models. Kinetic parameters characterizing peroxyl radical-scavenging activity showed that betanidin was more effective than betanin, in terms of both radical-scavenging rate constant and stoichiometric factor, with effectiveness of the same order as vitamin E under comparable conditions. Products identified by spectrophotometric and HPLC techniques indicated reaction of the glucose-substituted monophenol and ortho-diphenol moieties of betanin and betanidin, respectively, and suggested mechanisms of the antioxidant activity. Either betanin or betanidin incorporated in liposomes with α-tocopherol had additive effects, supporting partition of the pigments in the bilayer and lipoperoxyl radical reduction.     

Threshold effects on the lectin-mediated aggregation of liposomes: Influence of the diameter of the liposomes

Summary It had previously been found that small unilamellar liposomes of ca. 0.03 m diameter which bear synthetic cholesterol-containing glycolipids may be aggregated by an appropriate lectin [8]. Where studied, threshold effects have been observed in that the amount of glycolipid incorporated in the liposomes must exceed a certain minimum concentration in order for aggregation to occur [3, 8, 9, 13, 14]. Threshold effects of this type may be important in mediating cell-cell and virus-cell interactions. However, before studies with small unilamellar liposomes are useful as a model for these recognition and binding phenomena, it must be shown that the observed threshold effects are not associated with the very small radius of curvature of these liposomes. This article reports that larger liposomes of average diameter 0.26 and 0.45 m which contain the synthetic glycolipidl also show threshold effects when aggregated with the galactose binding lectin ricin agglutinin. Under conditions where more than 1% (mole) glycolipid is required to support the aggregation of the smallest liposomes, those of intermediate size require only 0.18% (mole) while the largest liposomes examined require between 0.095 and 0.15% (mole) depending on the method of preparation.     

The stabilization and release of hirudin from liposomes or lipid-assemblies coated with hydrophobically modified dextran

Hirudin is a 65-amino acid peptide and the most potent and specific known inhibitor of thrombin (K_i=0.2 pM). The short elimination half-life of hirudin from the body (1 hour) necessitates the use of a sustained and controlled delivery system. A proliposome method was used to entrap hirudin in liposomes coated with palmitoyl dextran-coated liposomes and lipid-assemblies. In vitro release studies of hirudin were performed using the lipid systems enclosed in dialysis membranes or deposited in the pores of a vascular graft. The activity of hirudin and released hirudin was measured using a thrombin chromogenic substrate assay.     

Interactions of liposomes with planar bilayer membranes

Summary The adhesion to horizontal, planar lipid membranes of lipid vesicles containing calcein in the aqueous compartment or fluorescent phospholipids in the membranes has been examined by phase contrast, differential interference contrast and fluorescence microscopy. With water-immersion lenses, it was possible to study the interactions of vesicles with planar bilayers at magnifications up to the useful limit of light microscopy. In the presence of 15 mM calcium chloride, vesicles composed of phosphatidylserine and either phosphatidylethanolamine or soybean lipids adhere to the torus, bilayer and lenses of planar bilayers of the same composition. Lenses of solvent appear, at the site where vesicles attach to decane-based bilayers and lipid fluorophores move from the vesicles to the lenses. Because the calcein contained in such vesicles is not released, we interpret this as indicating fusion of only the outer monolayer (hemifusion) of the vesicles with the decane lenses. In the case of squalene-based black lipid membranes (BLMs), in contrast, vesicles do not nucleate lenses but they apparently do fuse with the torus at the bilayer boundary. Interactions leading to hemifusions between vesicles and planar membranes thus occur predominantly in regions where hydrocarbon solvent is present. Osmotic water flow, induced by addition of urea to the compartment containing vesicles, causes coalescence of lenses in decane-based, BLMs as well as coalescence of the aqueous spaces of the vesicles that have undergone hemifusion with the lenses. We did not observe transfer of the aqueous phase of vesicles to therans side of either decane-or squalene-based planar membranes; however, we cannot rule out the possibility particularly in the latter case, that rupture of the planar membrane may have been an immediate result of vesicle fusion and thus precluded its detection.     

Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential

Among several promising new drug-delivery systems, liposomes represent an advanced technology to deliver active molecules to the site of action, and at present several formulations are in clinical use. Research on liposome technology has progressed from conventional vesicles ("first-generation liposomes") to "second-generation liposomes", in which long-circulating liposomes are obtained by modulating the lipid composition, size, and charge of the vesicle. Liposomes with modified surfaces have also been developed using several molecules, such as glycolipids or sialic acid. A significant step in the development of long-circulating liposomes came with inclusion of the synthetic polymer poly-(ethylene glycol) (PEG) in liposome composition. The presence of PEG on the surface of the liposomal carrier has been shown to extend blood-circulation time while reducing mononuclear phagocyte system uptake (stealth liposomes). This technology has resulted in a large number of liposome formulations encapsulating active molecules, with high target efficiency and activity. Further, by synthetic modification of the terminal PEG molecule, stealth liposomes can be actively targeted with monoclonal antibodies or ligands. This review focuses on stealth technology and summarizes pre-clinical and clinical data relating to the principal liposome formulations; it also discusses emerging trends of this promising technology.     

Preparation of DPPC liposomes using probe-tip sonication: Investigating intrinsic factors affecting temperature phase transitions

Liposomes are an important tool and have gained much attention for their promise as an effective means of delivering small therapeutic compounds to targeted sites. In an effort to establish an effective method to produce liposomes from the lipid, dipalmitoyl-phosphatidylcholine or DPPC, we have found important aspects that must be taken into consideration. Here, we used probe-tip sonication to prepare liposomes on a batch scale. During this process we uncovered interesting steps in their preparation that altered the thermodynamic properties and phase transitions of the resulting liposome mixtures. Using differential scanning calorimetry to assess this we found that increasing the sonication time had the most dramatic effect on our sample, producing almost an entirely separate phase transition relative to the main phase transition. This result is consistent with reports from the current literature. We also highlight a smaller transition, which we attribute to traces of unincorporated lipid that seems to gradually disappear as the total lipid concentration decreases. Overall, sonication is an effective means of producing liposomes, but we cannot assert this method is optimal in producing them with precise physical properties. Here we highlight the physical effects at play during this process.     

The Effect of Isoprene on the Properties of Spinach Thylakoids and Phosphatidyicholine Liposomes

Abstract: Isoprene is emitted from the leaves of some plants. It was recently reported that exogenous isoprene delays the onset of leaf damage during controlled increases in leaf temperature (Singsaas et al. Plant Physiology 115: 1413–1420 [1997^[17). Thylakoid membranes are presumed to be the site of action based upon isoprene's hydrophobicity, production in chloroplasts, and effect upon chlorophyll fluorescence at high temperatures. In an attempt to discern the mechanistic basis for isoprene's thermoprotective role, we studied the effect of exogenous isoprene on the peroxidation, permeability, and stability of spinach thylakoids and phosphatidylcholine liposomes. Isoprene, supplied at either 18 or 21 μ1 L¹, had no effect upon the rate of liposome peroxidation in the presence of a hydroxyl radical-generating system. Isoprene also did not affect liposome peroxidation at high temperatures. Neither the proton permeability of thylakoids nor the leakage of a fluorescent probe from liposomes was influenced by exogenous isoprene, when measured at several temperatures. Isoprene did not affect the stability of thylakoid membrane proteins during a temperature increase, as shown by differential scanning calorimetry. Therefore, despite the use of a variety of techniques to investigate fundamental membrane parameters, we were unable to demonstrate an effect of isoprene.     

Oligomeric state and membrane binding behaviour of creatine kinase isoenzymes: Implications for cellular function and mitochondrial structure

The membrane binding properties of cytosolic and mitochondrial creatine kinase isoenzymes are reviewed in this article. Differences between both dimeric and octameric mitochondrial creatine kinase (Mi-CK) attached to membranes and the unbound form are elaborated with respect to possible biological function. The formation of crystalline mitochondrial inclusions under pathological conditions and its possible origin in the membrane attachment capabilities of Mi-CK are discussed. Finally, the implications of these results on mitochondrial energy transduction and structure are presented.     

Fusion of influenza to liposomes is not inhibited by aliphatic primary alcohols

Reports of the antiviral activity of aliphatic alcohols led us to investigate the effects of aliphatic alcohols, from 10 to 20 carbons in length, on the phase transition behaviour of model phospholipids and on the fusion of influenza to liposomes. Contrary to the effects of many other antiviral agents, we find that alcohols are potent promoters of the inverted hexagonal phase. However, we also find that aliphatic alcohols have little effect on influenza fusion to liposomes. Eicosanol is the only aliphatic alcohol tested which substantially increases in fusion of influenza virus. We also find that long chain alcohols display multi-component bilayer to hexagonal phase transitions at higher mole fractions. This suggests that eicosanol may be facilitating fusion by creating defects between alcohol-rich and alcohol-poor regions of the lipid bilayer.     

Effect of anions on the cation selectivity of gramicidin-containing liposomes

Summary An osmotic method was used to study the salt permeability induced by gramicidin A in liposomes. Sequences of cation permeation were obtained for iodide, salycilate, acetate und formate salts in liposomes below and above their transition temperature. Salycilate and formate salts, unlike acetate and iodide salts, exhibit the same sequences for cation selectivity in liposomes below and above their transition temperature. These results can be explained by assuming three mechanisms for salt permeation across gramicidin-containing liposomes: (i) the anion moves by the lipid part of the membrane whereas the cation moves by the gramicidin channel, (ii) movement of the undissociated acid species occurs through the lipid part of the membrane followed by cation-proton exchange via the gramicidin channel and (iii) the cation and anion may move simultaneously via the gramicidin channel.When the movement of the anion or undissociated acid across the lipid part of the membrane is not rate limiting the permeation process, the cation selectivity obtained agrees with the cation selectivity of the gramicidin A channel, as determined by others using independent measurements.     

Enhancement of apparent substrate selectivity of proteinase K encapsulated in liposomes through a cholate-induced alteration of the bilayer permeability

Proteinase K-containing liposomes with highly selective membrane permeability properties were prepared. The selectivity obtained was with respect to the two substrate molecules added to the external aqueous phase of the liposomes: acetyl-L-Ala-Ala-Ala-p-nitroanilide (Ac-AAA-pNA) and succinyl-L-Ala-Ala-Ala-p-nitroanilide (Suc-AAA-pNA). The liposome-forming lipid used was POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and modulation of the membrane permeability was achieved using the detergent cholate. Proteinase K-containing mixed liposomes (PKCL) were prepared by adding cholate to preformed proteinase K-containing POPC liposomes (PKL) at a defined effective cholate/POPC molar ratio in the liposomal bilayer membrane R(e). Proteinase K was kept inside PKCL with a negligible amount of leakage into the bulk aqueous phase at R(e) < or = 0.30. At higher R(e), leakage of proteinase K was pronounced, even under conditions where POPC/cholate mixed liposomes seemed to be still intact (0.30 < R(e) < or = 0.39). At R(e) < or = 0.30, the reactivity of proteinase K in the PKCL measured with the externally added substrate Ac-AAA-pNA increased with increasing R(e), while the reactivity measured with Suc-AAA-pNA remained low, regardless of the R(e) value. This showed that externally added Ac-AAA-pNA molecules permeated the liposomal membrane more easily than Suc-AAA-pNA by modulating the membrane with cholate. Consequently, Ac-AAA-pNA was hydrolyzed in PKCL with considerably higher apparent substrate selectivity in comparison with the cases of proteinase K in PKL and free proteinase K (without liposomal encapsulation). The results obtained clearly demonstrate that the prepared PKCL can be utilized as a kind of nano-scaled bioreactor system which can take up a particular target substrate with high apparent substrate selectively from the external phase of the liposomes. Inside the liposomes, the target substrate is then converted into the corresponding products.     

T-cell antigen receptor triggering and lipid rafts: a matter of space and time scales. Talking Point on the involvement of lipid rafts in T-cell activation

T-cell antigen receptor triggering mechanisms and lipid rafts are of broad interest, but are also controversial topics. Here, we review some recent progress in these two research fields, which has been accomplished mostly in live cells and with the use of advanced technologies. We then discuss the potential relationship between membrane-domain organization and T-cell antigen receptor-triggering mechanisms. On the basis of the relevant experimental observations, we argue that the key to achieving a better understanding of both processes is the ability to monitor the molecular dynamics and interactions taking place in the membrane of T cells at a spatial scale of tens to hundreds of nanometres, with a subsecond-to-second temporal resolution.