Effects of Triton X-100 concentration and incubation temperature on carboxyfluorescein release from multilamellar liposomes

Carboxyfluorescein is the most commonly used probe to measure the rate of release of vesicle contents. The validity of the data obtained by this method depends on obtaining an end point based on the complete release of the dye on treatment of the liposomes with a detergent, usually Triton X-100. However, Triton does not completely release entrapped carboxyfluorescein from multilamellar liposomes and the amount and rate of release of marker upon detergent treatment is a function of lipid composition of the liposome, Triton concentration and temperature and duration of detergent incubation. The fluorescence ‘end point’ for distearoyl-l-α-phosphatidylcholine/cholesterol (2:1, mol%) multilamellar liposomes treated with 0.5% Triton at 22°C (a condition often used) is only about one-fifth the value for liposomes treated with 5% Triton at 72°C. The conditions of treatment appear to affect the release of carboxyfluorescein from the lipid of the partially or completely disrupted liposome and the subsequent partitioning of the free dye into the aqueous phase. This effect can lead to serious errors in the interpretation of multilamellar liposome stability data. However, Triton allows complete release of entrapped dye from small unilamellar vesicles under all conditions tested.     

Radioprotective effects of lipid A, liposomes, and liposomes containing lipid A in mice

Lipid A from Gram-negative bacterial lipopolysaccharide (endotoxin) was incorporated into liposomal membranes and examined as a prophylactic radioprotectant compound in lethally irradiated mice. Splenic hematopoietic activity, resulting in increased numbers of spleen cell colonies, was induced both by lipid A alone or more strongly by liposomal lipid A. Increased survival of lethally irradiated animals was induced to a slight extent by liposomes alone, to a greater extent by lipid A, and at the highest level by liposomes containing lipid A. Under conditions where 100% of untreated or saline-treated animals died of acute radiation syndrome after 20 days, more than 90% of the animals pretreated with liposomal lipid A were still alive 30 days after irradiation. We conclude that lipid A had substantial radioprotectant activity by itself, and the activity was enhanced by incorporation into liposomes. Liposomes alone also exhibited mild radioprotectant effects.     

Partitioning and membrane disordering effects of dopamine antagonists: influence of lipid peroxidation, temperature, and drug concentration.

The effect of four dopamine antagonists (spiperone, haloperidol, pimozide, and domperidone) on the lipid order of caudate nucleus microsomal membranes and on liposomes from membrane lipid extracts was evaluated and related to the partition coefficients (Kp) of the drugs. Lipid membrane order was determined by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe of the membrane core and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a probe of the membrane surface. Dopamine antagonists decrease the fluorescence polarization of both probes, indicating that they disorder the membrane lipids at different depths. Pimozide and domperidone, the drugs with higher Kp values, are more effective at decreasing the polarization of DPH, a probe of the membrane core, than that of TMA-DPH. In contrast, spiperone and haloperidol, which have lower values for Kp, induce more significant decreases in TMA-DPH depolarization, a probe of the membrane surface. These findings indicate that higher partition coefficients of the drugs are directly correlated with an increase of fluidity in the hydrophobic core of brain membranes. Ascorbate/Fe(2+)-induced membrane lipid peroxidation increases membrane order. Membrane lipid peroxidation decreases the partition coefficients of the dopamine antagonists tested. Increasing temperature (4-37 degrees C) decreases membrane order, but temperature effect is less evident after lipid peroxidation. The disordering effect of dopamine antagonists increases with increasing drug concentrations (1-15 microM), a maximum being observed at 10 microM. However, this effect is also less evident after membrane lipid peroxidation. We can conclude that dopamine antagonists and membrane lipid peroxidation affect membrane lipid order and that the action of these drugs is dependent on initial bilayer fluidity. Membrane lipid peroxidation increases membrane order while dopamine antagonists show a disordering effect of membrane phospholipids. This disordering effect can indirectly influence the activity of membrane proteins and it is one of the mechanisms through which membrane function can be altered by these drugs.     

Nitrogen release from decomposing seaweeds: species and temperature effects

This study determined the rate at which nitrogen accumulated in seaweeds is released during decomposition and the effect of temperature on their rates of decomposition and nitrogen release. Gracilaria verrucosa and Ulva lactuca decomposed rapidly in outdoor mesocosms. Ulva, but not Gracilaria, became nitrogen-enriched during decomposition. Maximal weekly rates of nitrogen release were 5.91 ± 2.23 and 6.37 ± 2.59 g N m^?2 d^?1, respectively for Gracilaria and Ulva. Temperature had a significant effect on the decomposition rate of Gracilaria in a laboratory experiment: decomposition was greater at 30 °C than at 25 °C. No net decomposition was observed at 16 °C. Gracilaria became nitrogen enriched at 30 °C, but not at 16° or 25°. The release of stored nutrients from decaying seaweeds should be included in nutrient budgets and models when seaweed standing stocks are significant. Seaweed source-sink relationships are important ecologically and can be applied to attempts at using seaweeds as environmental monitors of anthropogenic eutrophication and to efforts of cultivating seaweeds for the improvement of water quality.     

Pheromone release by the two forms of Callosobruchus maculatus females: effects of age, temperature and host plant

Abstract. The influence of age, temperature and host plant on pheromone release was investigated in females of two forms of Callosobruchus maculatus (Coleoptera: Bruchidae), referred to as the flightless form and the flight form. The emission of pheromone seemed to be correlated with reproductive status: the flightless-form females which were sexually mature at emergence began to release pheromone on the first day following emergence. The flight-form females which exhibited no reproductive activity at emergence also showed a delayed emission of pheromone. The temperature conditions and the availability of host plant seeds had no apparent effect on pheromone release by the flightless form. In flight-form females pheromone release was stimulated by the presence of host plant seeds. Temperature seemed to affect the age at which females initiated pheromone release for the first time after emergence, pheromonal emission being brought forward slightly by higher temperature. All these results are discussed relative to the specific environment of each form.     

Parameters affecting fusion between liposomes and synaptosomes. Role of proteins,lipid peroxidation,pH and temperature

We investigated the effect of several parameters, such as temperature, pH and proteins, on the fusion between synaptosomes, freshly isolated from rat brain cortex, and large unilamellar phosphatidylserine liposomes. These studies were carried out in both peroxidized and nonperoxidized synaptosomes. Mixing of membrane lipids was monitored using a fluorescence resonance energy transfer assay. Ascorbate (0.8 mm)/ Fe²⁺ (2.5 m)-induced peroxidation of synaptosomes enhanced the fusion process (twofold) which may reflect an increase in synaptosomal protein hydrophobicity and hence a facilitation of intermembrane aggregation. The fusion process was shown to be temperature sensitive, a reduction in the extent being observed (twofold) as the temperature was lowered from 37 to 25°C. This effect may be due to changes in membrane fluidity. The fusion process is pH dependent, an increase in both kinetics and extent being observed when the pH was lowered from 7.4 to 5.5. A significant inhibition (92% at pH 7.4; 35% at pH 5.5) of the interaction between synaptosomes and liposomes by trypsin pretreatment of synaptosomes was found, thus indicating that the fusion reaction is a protein-mediated process. The inhibitory effect of trypsin at pH 5.5 is not so strong as that at physiological pH. These results suggest that, in addition to the involvement of proteins, nonspecific interactions between the synaptosomal and liposomal membranes under acidic conditions may also play a role in the fusion process. The investigation of binding of synaptosomes to liposomes under several experimental conditions provided evidence for the participation of proteins in membrane aggregation, as well as for the role of electrostatic forces in this process, at mild acidic pH.This work was supported by Junta National de Investigação Científica e Tecnológica (JNICT) and the Calouste Gulbenkian Foundation, Portugal.     

Reorientational dynamics in lipid vesicles and liposomes studied with ESR: effects of hydration, curvature and unsaturation

Electron spin resonance experiments were carried out on 3-doxyl-5 alpha-cholestane spin-label (CSL) molecules embedded in multilamellar liposomes and small unilamellar vesicles (SUVs) of palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC) and dilinoleoylphosphatidylcholine (DLPC). The experimental spectra were analyzed by a numerical solution of the stochastic Liouville equation. Effects of temperature, presence of unsaturated bonds and high bilayer curvature on the dynamic behaviour of the lipid molecules were studied. Our results, combined with results from planar multibilayers with a varying hydration rate (Korstanje et al. (1989) Biochim. Biophys. Acta 980, 225-233), give a consistent picture of the orientational order and rotational dynamics of CSL molecules embedded in lipid matrices with various geometrical configurations. Increase of hydration or temperature reduces molecular ordering and increases molecular dynamics. In highly curved vesicle configurations, SUVs, molecular order is found to be lower than in multilamellar liposomes. In contrast, rotational motion is not affected by increase of curvature. In all lipid configurations studied, increase of the number of unsaturated bonds in the fatty acid chains reduces molecular ordering. We find, however, no effect of unsaturation on the rotational mobility of the CSL probe molecules. These results clearly show that changes in molecular orientational order and reorientational dynamics have to be considered separately, and that they are not necessarily correlated as implied by the common concept of membrane fluidity. Comparing our results with data from a motional narrowing analysis shows that the latter approach seriously overestimates the rate of molecular reorientation.     

The effects of temperature,salinity, concentration and PEGylated lipid on the spontaneous nanostructures of bicellar mixtures

The self-assembling morphologies of low-concentration (mostly 1 and 10 mg/mL) bicellar mixtures composed of zwitterionic dipalmitoyl (di-C₁₆) phosphatidylcholine (DPPC), dihexanoyl (di-C₆) phosphatidylcholine (DHPC), and negatively charged dipalmitoyl (di-C₁₆) phosphatidylglycerol (DPPG) were investigated using small angle neutron scattering, dynamic light scattering and transmission electron microscopy. A polyethylene glycol conjugated (PEGylated) lipid, distearoyl phosphoethanolamine-[methoxy (polyethyleneglycol)-2000] (PEG2000-DSPE), was incorporated in the system at 5 mol% of the total lipid composition. The effects of several parameters on the spontaneous structures were studied, including temperature, lipid concentration, salinity, and PEG2000-DSPE. In general, nanodiscs (bicelles) were observed at low temperatures (below the melting temperature, T_M of DPPC) depending on the salinity of the solutions. Nanodisc-to-vesicle transition was found upon the elevation of temperature (above T_M) in the cases of low lipid concentration in the absence of PEG2000-DSPE or high salinity. Both addition of PEG2000-DSPE and high lipid concentration stabilize the nanodiscs, preventing the formation of multilamellar vesicles, while high salinity promotes vesiculation and the formation of aggregation. This study suggests that the stability of such nanodiscs is presumably controlled by the electrostatic interactions, the steric effect induced by PEG2000-DSPE, and the amount of DHPC located at the disc rim.     

Stimulation of ion release from liposomes by the polyene antibiotic, filipin.

The effect of the polyene antibiotic, filipin, upon release of the ions Ca²⁺, Sr²⁺, SO₄^2? and phosphate out of phospholipid and phospholipid-cholesterol liposomal vesicles was studied. The addition of filipin at concentrations stoichiometrically comparable to the cholesterol concentration in the liposomes, resulted in 2–10 × stimulation of the rate of release of all of these ions. The filipin mediated stimulation of release of ions from liposomes was dependent upon the presence of cholesterol. The relative effectiveness of filipin increased when the mole percent of cholesterol incorporated into the liposomes increased from 10 to 50% and when the molar filipin:cholesterol ratio increased from 0.2 to 1.0. It has been previously shown that there is a 1:1 stoichiometry of interaction between filipin and cholesterol [Biochem. Biophys. Acta339, 57 (1974)]. The present studies suggest that this 1:1 stoichiometric interaction may also be responsible for the increased release of entrapped ions.A possible mechanism of action of polyene antibiotics is discussed which suggests that the rearrangement of membrane constituents occurring upon interaction of filipin with cholesterol is the basis for the enhancement of ion release. This would imply that the ion specificity observed upon interaction of polyene antibiotics with membranes would not only be determined by the polyene antibiotic itself, but also by the intrinsic properties of the membrane.     

Photoisomerisable cholesterol derivatives as photo-trigger of liposomes: effect of lipid polarity, temperature, incorporation ratio, and cholesterol

Three cholesterol derivatives containing an azobenzene moiety with different polarities were designed and synthesized (AB lipids 1 to 3). The effects of structure, temperature and incorporation ratio on liposomes were studied, with the results showing that the polarity in 4-substituent and in some cases, 4'-substituent may be important for their incorporation feasibility and photoisomerizability in liposomes. Liposomes incorporated with AB lipid 3 could release multi-pulsatilely upon UV and visible light irradiation both in gel state and liquid crystal state of liposomes. An increase in the incorporation ratio of AB lipid 3 enhanced the amount of drug released greatly. Unlike other azobenzene photo-triggers reported, AB lipid 3 did not increase the spontaneous release of liposomes. Furthermore, cholesterol suppressed the spontaneous release of liposomes.     

Binding of insulin to the external surface of liposomes: Effect of surface curvature,temperature, and lipid composition

The binding of insulin to the external surface of phosphatidylcholine liposomes as a function of the temperature, the surface curvature, and the composition of lipids was studied. The amount of the saturated binding of insulin to liposomes was assessed by gel-filtration chromatography. The binding of insulin to small unilamellar vesicles was highly dependent upon the temperature, favoring low temperatures. As the temperature increased, there was a distinct temperature range where the binding of insulin to small unilamellar vesicles decreased. The temperature ranges for dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles were found to be 10–20°C and 21–37°C, respectively. These temperature ranges were quite different from the reported ranges of the gel → liquid crystalline phase transition temperatures ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) for DMPC or DPPC small unilamellar vesicles. In contrast to other proteins, the amount of insulin bound to DMPC and DPPC small unilamellar vesicles was negligible at or above the upper limit of the above temperature ranges, and increased steadily to 6–7 μmol of insulin per mmol of phospholipid as the temperature decreased to or below the lower limit of these temperature ranges. On the other hand, the binding of insulin to the large multilamellar liposomes cannot be detected at all temperatures tested. The affinity of insulin to neutral phosphatidylcholine small unilamellar vesicles appeared to be related to the surface curvature of the liposomes, favoring the liposomes with a high surface curvature. Furthermore, the amount of insulin bound to small unilamellar vesicles decreased as the content of the cholesterol increased. The presence of 10% molar fraction of phosphatidic acid did not appear to affect the binding of insulin to small unilamellar vesicles. However, the presence of 5% molar fraction of stearylamine in DPPC small unilamellar vesicles increased the amount of bound insulin as well as the extent of aggregation of liposomes. The results of the present study suggest that the interstitial regions of the acyl chains of phospholipids between the faceted planes of small unilamellar vesicles below $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ may be responsible for the hydrophobic interaction of insulin and small unilamellar vesicles. The tight binding of insulin to certain small unilamellar liposomes could lead to an overestimation of the true amount of insulin encapsulated in liposomes, if care is not taken to eliminate the bound insulin during the procedure of encapsulating insulin in liposomes.     

Interactive effects of salt concentration and temperature on growth and lipid composition in the moderately halophilic bacterium Vibrio costicola.

The interactive effects of NaCl concentration and growth temperature on the growth and lipid composition of the moderately halophilic eubacterium Vibrio costicola have been investigated. Vibrio costicola was shown to be capable of growth over the temperature range 4-37 degrees C. Maximum growth yields were obtained at 30 degrees C when the optimum NaCl concentration was 1.0 M NaCl. In contrast with some previous studies, at higher or lower growth temperatures both the optimum and lower limit of NaCl concentration were higher, but there was no change in the upper limit of NaCl concentration for growth. There were no differences between the lipid compositions of cultures grown in 1 M NaCl at 30 or 37 degrees C, but as the growth temperature was lowered from 30 to 10 or 4 degrees C, the ratio of phosphatidylethanolamine to phosphatidylglycerol increased significantly as a result of the conversion of phosphatidylglycerol to diphosphatidylglycerol; in addition, at the lower growth temperatures the phospholipid fatty acyl composition became more unsaturated and the mean acyl chain length was shorter. It is suggested that the altered salt dependence of V. costicola at temperatures below the optimum for growth is due to a modification in membrane lipid phase behavior and stability brought about by changes in lipid composition, whereas a different mechanism operates above the growth temperature optimum.     

Comparison of dynamics of lecithin liposomes and brain total lipid liposomes with synaptosomal membranes. An EPR spectroscopy study.

Dynamics and/or order of the hydrophobic part of phosphatidylcholine (PC) liposomes and rat brain total lipid (TL) liposomes and synaptosomes were studied and compared by EPR spectroscopy using the spin probes 5 or 16-doxyl stearic acid and 14-doxyl phosphatidylcholine. The dynamics and/or order of the hydrophobic part of TL liposomes or synaptosomes were similar but differed largely from those of PC liposomes. The dynamics of the hydrophobic part of the liposomes decreased gradually with the increasing TL/PC ratio in the sample. To obtain in TL liposomes or synaptosomes the same EPR spectrum parameters as in PC liposomes at 37 degrees C, the formers have to be heated to temperatures of approximately 50-60 degrees C. The dynamics and/or order of the hydrophobic part of lecithin liposomes at 5-10 degrees C were comparable with those of TL liposomes or synaptosomes at 37 degrees C. The results emphasize the role of the lipid composition in studies concerning drug-lipid and protein-lipid interactions in model and biological membranes.     

Effect of temperature and surfactant on the control release of microencapsulated dye in lecithin liposomes. I

The objective of our work has been the microencapsulation of dyes with lecithin from soybean, with the formation of liposomes, as a substitute for synthetic auxiliaries so as to improve the quality of the effluent. Current scenarios promote the disintegration and leakage of the liposomes, such as, changes in temperature, pH and the use of surfactants. Since dyeing process is a mix of all these parameters, we pretended to study each one separately. Rhodamine 6G fluorescence is known to be concentration quenched through the formation of non-fluorescent dimmers and, additionally, through the energy transfer from rhodamine monomer to these dimmers (Baptista ALF, Coutinho PJG, Real Oliveira MECD, Gomes JINR. Proceedings of 13th International Symposium of Surfactants, SIS 2000, Gainesville, USA, 2000). The temperature, the surfactant and pH induce a release of the encapsulated dye resulting in rhodamine dilution and consequently alterations in the dimerization/binding equilibrium. The experimental spectra indicate that rhodamine binds almost completely to liposomes. The decomposition of the rhodamine fluorescence spectra allowed us to determine the percentage of released dye during a simulated dyeing process, and allowed us to conclude that the dimerization process occurs mainly at the inner interfaces. The amount of dye released induced by temperature changes was greater in the presence of surfactant.     

Studies on the compartmentation of lipid in adipose cells. I. Subcellular distribution, composition, and transport of newly synthesized lipid: liposomes

The subcellular distribution and composition of endogenously synthesized lipid in isolated white adipose cells were studied to determine the nature and extent of lipid compartmentation. After brief incubation of cells with labeled glucose, acetate, or palmitic acid, over 90% of newly synthesized triglyceride was localized in the bulk-lipid phase, indicating rapid intracellular transport and storage. From 13 to 20% of the newly formed lipid was diglyceride, and over 95% of it was localized in the central lipid-storage vacuole rather than in organelle systems concerned with esterification, thus indicating intracellular segregation of newly synthesized partial glycerides. Most of the newly synthesized phosphatides partitioned with membranous organelles. Synthesis of cholesterol or cholesteryl ester was negligible. After brief incubation of cells with labeled glucose, the relative specific activity of organelle triglyceride was mitochondria > microsomes > liposomes > soluble supernatant > bulk lipid. In pulse-chase studies the specific activity of organelle triglyceride decreased and that of the bulk fraction increased reflecting intracellular lipid transport. The data suggest that a significant proportion of newly formed lipid is transferred from mitochondrial membranes into the storage vacuole by direct lipid-lipid interaction. Liposomes, which consist of small enclosed lipid droplets resembling chylomicrons, contained triglycerides of specific activity similar to microsomal triglyceride. While the evidence that liposome triglyceride may be microsomal in origin is indirect, the results do indicate that the liposome fraction represents a phase in the transport and(or) storage of new glyceride. At least two forms of compartmentation of newly synthesized lipids occurred. The first, termed "structural," refers to localization of lipids to organelle fractions. The second type of compartmentation, termed "chemical," concerns the intracellular segregation of a specific lipid class. The accumulation and segregation of newly synthesized diglyceride in the bulk storage pool are examples of the latter form of compartmentation.     

Molecular diffusion into ferritin: pathways, temperature dependence, incubation time, and concentration effects

The detailed kinetics of permeation and effusion of small nitroxide spin probe radicals with the protein shells of horse spleen ferritin (HoSF) and human H-chain ferritin (HuHF) and a 3-fold channel variant D131H+E134H of HuHF were studied by electron paramagnetic resonance spectroscopy and gel permeation chromatography under a variety of experimental conditions. The results confirm that the permeation of molecular species of 7-9-A diameter into ferritin is a charge selective process and that the threefold channels are the likely pathways for entry into the protein. Studies with holoHoSF show that increased temperature increases the rates of penetration and effusion and also increases the concentration of positively charged spin probe accumulated within the protein in excess of that in the external solution. The interior of HoSF is much more accessible to small molecules at physiological temperature of approximately 40 degrees C than at room temperature. The large activation energy of 63-67 kJ/mol measured for the effusion/penetration and the small diffusion coefficient, D approximately 5 x 10(-22) m(2)/s at 20 degrees C, corresponding to a time of approximately 60 min for traversing the protein shell, is consistent with the kinetics of diffusion being largely controlled by the restrictive porosity of the protein itself. An inverse dependence of the first-order rate constant for effusion out of the protein channel on the incubation time used for radical penetration into the protein is attributed to increased binding of the radical within the funnel-shaped channel.     

Phospholipid vesicle fusion and drug loading: temperature,solute and cholesterol effects,and, a rapid preparation for solute-loaded vesicles

The fusion of sonicated dipalmitoylphosphatidylcholine (DPPC) vesicles was studied by gel-exclusion chromatography as a function of temperature, permeable and impermeable solute concentration, and cholesterol content of the bilayer membrane. Fusion is faster at lower temperatures: there is no fusion at or above 35.5°C (0.10 M DPPC/0.1 M K₂SO₄/0.01 M Hepes buffer (pH 7.4)/0.02% NaN₃). There is about 10% fusion after 1 week at 30°C, and about 60% fusion after 2 days at 13–25°C. Between 13 and 8°C, the fusion product changes from 700-Å-diameter vesicles to the 950-Å vesicles previously reported by Wong et al. (Wong, M., Anthony, F.W., Tillack, T.W. and Thompson, T.E. (1982) Biochemistry 21, 4126–4132). At 1°C, fusion is about 90% complete after 1 day. Membrane-impermeable solutes (NaCl, trehalose and glucose) inhibit fusion in a manner reflecting the total particle concentration. There is no detectable fusion after 3 days (22°C) in either 1.0 M NaCl or 2.0 M sugar, the highest concentrations studied. A suggested explanation is that impermeable solutes osmotically inhibit the influx of solution that accompanies the fusion of vesicles to form a larger vesicle, and, could conceivably thereby inhibit the fusion reaction. By contrast, membrane-permeable solutes (glycerol, ethylene glycol, propylene glycol and ethanol) dramatically increase the fusion rate. 1.0 M ethanol causes 100% fusion in 15–30 min at 22°C. The simultaneous presence of 0.15 M NaCl entirely negates the fusion-promoting effect of 1.0 M ethanol. 1 mol% cholesterol completely inhibits fusion in 0.1 M KCl (20°C), and greatly slows it down either in 1.0 M ethanol at 20°C or in 0.1 M KCl at 4°C. A suggested mechanism is that cholesterol might concentrate in and stabilize bilayer lattice defect sites that are critical for the fusion reaction. The trapping efficiency of vesicles formed by the fast ethanol fusion conditions in the presence of the water-soluble markers, chromate and arsenazo III, ranged from 9.0 to 12.7% of the marker captured in the vesicles, corresponding to trapped volumes of 1.8 to 2.5 1/mol DPPC. Bromophenol blue gave anomalously high values of 67% and 13 1/mol DPPC, which presumably reflect binding, in addition to encapsulation.