Effect of carbohydrates and glycerol on the stability and surface properties of lyophilized liposomes

Summary The interaction of permeant molecules such as glycerol and urea and nonpermeants such as trehalose, sucrose, lactose and glucose with dipalmitoylphosphatidylcholine (DPPC) and egg yolk phosphatidylcholine (EPC) bilayers was studied by means of infrared spectroscopy in solid samples. The properties of the liposomes formed upon rehydration in different polyol solutions were determined by dynamic light scattering, fluorescence anisotropy, absorbance at 450 nm and merocyanine 540 spectra.Phospholipid samples dehydrated in the presence of urea and glycerol give v _1/2 values for the antisymmetric stretch (P=0 stretch) in the IR spectra; lower values are found for hydrated phospholipids.In contrast, the same procedure in the presence of carbohydrates, gives v _1/2 values close or higher to those found for hydrated phospholipids, following the sequence glucose>sucrose>trehalose. This order is similar to that found in hydrated bilayers for the 570/500 nm ratio determined in the MC 540 spectra as a function of the number of OH equatorial groups of the sugars.Liposomes lyophilized in the presence of those carbohydrates and rehydrated in buffer solution showed an increase in the 570/530 absorbance ratio in the MC spectra at temperatures below that corresponding to the gel-liquid crystalline transition. This is interpreted as an exposure of hydrophobic regions due to the carbohydrate-phospholipid interaction. In these conditions, the size at which liposomes spontaneously stabilize is a function of the type and concentration of the polyols in the aqueous solution. These changes in size are connected with packing and mechanical constraints of the bilayer for some of the sugars assayed.Similar results to those obtained with lyophilized liposomes were found after aging liposomes in high sugar concentration solutions. A clear distinction can be made between the effect of permeant and nonpermeant molecules in regard to size, packing and hydrophobic region exposure.     

Surface changes induced by osmotic stress and its influence on the glycerol permeability in lipid bilayers

The penetration rate of glycerol across lipid bilayers can be assayed dispersing liposomes filled with a 0.1 M glucose solution in an isotonic or a hypertonic solution of glycerol. The kinetic of glycerol permeation is found to be different in each of those cases. Liposomes dispersed above the phase transition temperature in hypertonic solutions show an increase in the surface polarization as measured by means of merocyanine 540. Under this condition, the permeation of glycerol shows a two-step kinetic which is indicative of a non-fickean diffusion process. In contrast, liposomes dispersed in isotonic solutions of the permeant show a fickean behavior. The changes in polarization of the membrane interface are ascribed to variations in the surface potential due to the osmotic collapse and the glycerol concentration in contact with the outer surface. The permeability of polar molecules can, in consequence, be considered as a function of the surface potential of the liposome which is congruent with previous data in literature reporting that water permeability increases as a function of the zeta potential of liposomes shrunken in hypertonic solutions.     

Senescence-Dependent Increase in the Permeability of Liposomes Prepared from Bean Cotyledon Membranes

Liposomes containing 79 mM Tris-acetate and 50 mM KCl were preparedfrom the total lipid extracts of smooth microsomal membranesisolated from 2, 4, 7 and 9 d old bean cotyledons. Permeabilityto glycerol was determined by spectrophotometric measurementsof osmotic swelling when the liposomes were placed in eitherisotonic or slightly hypotonic glycerol. For liposomes from2 and 4 d old membrane there was a time-dependent decrease inabsorbance at 450 nm from which initial swelling rates reflectingthe influx of glycerol and water were calculated. At 25 °Cthese rates were not significantly different For liposomes from7 and 9 d old membrane there was no change in absorbance withtime at 450 nm signifying that these older liposomes were equallypermeable and non-electrolytes and non-electrolytes, and thereforeincapable of swelling. Permeability to glucose was determinedby preparing the liposomes in a solution of the sugar, passingthem through a Sephadex column to eliminate unsequestered glucose,and quantifying sugar leaked from the liposomes over time bymeasuring NADPH formation through the tandem actions of hexokinaseand glucose-6-phosphate dehydrogenase. The rate constants forglucose leakage from 2 and 7 d old liposomes were 0.55 and 3.94respectively, again indicating a dramatic increase in permeabilitywith advancing age. These changes in permeability correlatetemporally with the appearance of gel phase lipid in both liposomesand the membranes from which they were derived, suggesting thatthe coexistence of discrete liquid-crystalline and gel phaselipid domains renders membranes leaky and contributes to lossof intracellular compartmentation in senescing tissue.     

Membrane-potential- and surface-potential-induced absorbance changes of merocyanine dyes added to chromatophores from Rhodopseudomonas sphaeroides

(1) Three analogs of merocyanine dyes added to suspensions of chromatophore vesicles showed absorbance changes responding to the change in surface potential induced by salt addition and to the change in membrane potential induced by illumination. (2) The extent of the light-induced absorbance changes of the dyes was linearly related, in the presence and absence of uncouplers, to that of carotenoid spectral shift which is an intrinsic probe of the intramembrane electric field. (3) Comparison of the merocyanine absorbance changes induced by salt addition with those induced by illumination indicated that the surface potential change in the outer surface of chromatophore membranes during illumination was very small. (4) Judging from the spectra of these absorbance and from the low permeabilities of the dyes to membrane, the absorbance change are attributed to change in distribution of the dyes between the medium and the outer surface region in chromatophore membranes. The extent of the light-induced absorbance changes of merocyanine dyes depended on the salt concentration of the medium. The types of dependence were different among three merocyanine analogs. This is explained by the mechanism mentioned above assuming appropriate parameters. It is suggested that, under continuous illumination, an equilibrium of the electrochemical potential of H⁺ is reached between the bulk aqueous phase and the outer surface region in the membrane where the merocyanine dyes are distributed.     

Temperature dependence of optical properties of chlorophyll a incorporated into phosphatidylcholine liposomes

Temperature-dependent spectral changes of chlorophyll a (Chl a) incorporated into liposomes of two types of phosphatidylcholine are studied. When Chl a incorporated into the liposomes is cooled down to 5°C from the temperature of the gel-to-liquid crystalline phase transition of the lipid, the red shift as well as the increase in half-bandwidth of the red peak of Chl a are only slight. By measuring the difference spectra produced by substracting the absorption spectrum at the phase transition temperature of the lipid from that at lower temperature, it is shown that the component absorbing at longer wavelength (675–685 nm) than the peak of the red maximum (about 670 nm) significantly increases at the expense of the component absorbing at shorter wavelength (657–668 nm). The positions of positive and negative peaks depend on the temperature and the molar ratio of the lipid to Chl a. The absorbance change is most pronounced on cooling below the phase transition temperature of the lipid. The temperature-induced absorbance change is almost completely reversible. The results indicate that the aggregated forms of Chl a in liposomes can be spectrophotometrically detected in the gel phase of the lipid.     

Spectral changes in membrane fragments and artificial liposomes of Anacystis induced by chilling.

Isolated membrane fragments from Anacystis nidulans grown at 39 °C undergo visible spectral changes on chilling, suggesting a carotenoid component is altered. No such changes are seen when cells are grown at 25 °C. The magnitude of the decreased absorbance is a function of the chilling temperature and the media in which membrane fragments are suspended. The spectral decrease following chilling develops relatively slowly and is a function of the cooling rate and final temperature. The absorbance change is reversed if the fragments are heated to near 50 °C subsequent to chilling. Liposomes prepared from a total lipid extract of Anacystis undergo a spectral change on chilling which closely resembles that occurring in whole cells or isolated membrane fragments. Liposomes prepared from an extract of cells grown at 25 °C show only about 30% as great a spectral change as those from cells grown at 39 °C. The spectral bleaching is freely reversible when the liposomes are reheated, but shows a pronounced hysteresis. It is suggested that specific phase changes occur in Anacystis membranes and artificial liposomes on cooling which alter the environment of carotenoid. These changes may relate to previous observations that cells grown at 39 °C cannot survive a cold shock while those grown at 25 °C do.     

Effect of membrane composition on lipid oxidation in liposomes

To study the effect of membrane composition on the oxidation of liposomes, different systems were prepared by adding one component at time to phosphatidylcholine (Epikuron 200). In particular, the effect of cholesterol and its ester, cholesterol stearate, on membrane structure and oxidation was studied. A first screening of the structure and net charge of the different preparation was made by means of z-potential and size measurements. Then the liposomes were oxidized by using a hydrophilic radical initiator, the (2,2-azobis(2-amidinopropane) hydrochloride, AAPH, which thermally decomposes to give a constant radical flux in water. The oxidation of liposomes, monitored by following the absorbance of the primary products of oxidation at 234 nm, was shown to be dependent on the composition of the liposomal bilayer and so on its biophysical properties. In addition, size and z-potential measurements gathered in the time course of the peroxidation reaction, revealed that the oxidation induced a modification of the superficial characteristics of the membrane bilayer so as to change its charge at the shear plane (z-potential). This behaviour was shared by all liposomal preparations independent of the composition. The change in sizes of the different liposomal preparation, instead, followed different trends, being more stable both in control samples and in oxidized ones when cholesterol was present. From the analysis of the results, it can be concluded that cholesterol affects the oxidation induced by hydrophilic radical initiator of model membranes by changing the biophysical properties of the phospholipid bilayer. The rigidity induced by cholesterol at temperatures above the T_m makes the membrane more resistant to radical attack from an external aqueous phase and this in turn delays the start of the reaction. The decrease of z-potential of the liposomal particles induced by the oxidation process can be an important clue to understand the mechanisms involved in the etiology of important diseases.     

Synthetic glycolipids and the lectin-mediated aggregation of liposomes.

Synthetic mannose-containing glycolipids utilizing the cholesterol nucleus as a lipid anchor, and either the 6-aminohexyl- or the 6-(6-aminohexanamido)hexyl-1-thio-alpha-D-mannopyranosides as the carbohydrate ligands, have been synthesized and incorporated into small unilamellar liposomes. Incorporation of these cholesterol-mannoside derivatives at concentrations up to 14 mol% apparently does not affect the physical characteristics of the liposomes. Addition of concanavalin A to a suspension of liposomes containing the long chain cholesterol-mannose derivative causes an increase in light-scattering at 360 nm. As the increase in absorbance is completely reversed by the addition of alpha-methylmannoside, aggregation rather than fusion of the liposomes appears to be occurring. Liposomes containing 14 mol % of the short chain (6-aminohexyl-) derivative are aggregated by concanavalin A indicating that the lectin can approach to within 10 A of the lipid bilayer. Preliminary results suggest that the aggregation of vesicles containing either the long or short chain derivatives is highly dependent on the density of the sugar in the membrane.     

Osmotic dependence of the lysophosphatidylcholine lytic action on liposomes in the gel state

Multilamellar liposomes of dimyristoylphosphatidylcholine are susceptible to lytic action of lysophosphatidylcholine at the gel state, an effect which is not observed when liposomes are in the liquid crystalline state. The lytic action has been found to be enhanced when liposomes are dispersed in hypertonic solutions. On the contrary, hypotonic solutions decreased the effectiveness of the lysolipid. Shrunken liposomes present surface changes as detected by merocyanine 540 and 1-anilinonaphthalene-8-sulfonic acid which can be ascribed to the spontaneous curvature promoted by shrinkage.     

Modifications to Lambert and Neish's method of glycerol determination

The sensitivity of the method for glycerol determination by Lambert and Neish (Lambert, M. and Neish, A.C. (1950) Can. J. Sect. B 28, 83–89) can be enhanced by simple modifications of the procedure. As much as a five-fold increase in the sensitivity of the method could be obtained by measuring the absorbance of the coloured complex at 367 nm instead of 570 nm and by prolonging the heating period for colour development.     

Membrane phase state and the rearrangement of hematopoietic cell surface receptors.

Transformed murine hematopoietic cells of several lineages bound the fluorescent membrane probe merocyanine 540, whereas their normal counterparts did not. Similar selective binding was reproduced in artificial liposomes which bound this probe above their phase transition temperature, but not below it. The regions of the membrane to which merocyanine 540 binds along with the receptors for the lectin concanavalin A, but not the receptors for the lectin wheat germ agglutinin, were rearranged during the course of induced differentiation of erythroleukemia cells. Based on these findings, we propose a model of hematopoietic cell surface differentiation in which proteins such as concanavalin A receptors, which are destined for removal from the plasma membrane, are specifically associated with disordered, liquid-like lipid domains which can be visualized with merocyanine 540. For the specific case of erythroid differentiation, these domains and their associated proteins are collected at the region of the membrane where nuclear extrusion occurs and are eliminated from the reticulocyte plasma membrane by the enucleation event.     

Bacteriorhodopsin photoreaction: identification of a long-lived intermediate N (P,R350) at high pH and its M-like photoproduct

An alkaline suspension of light-adapted purple membrane exposed to continuous light showed a large absorption depletion at 580 nm and a small increase around 350 nm. We attribute this absorption change to an efficient photoconversion of bR570 into a photoproduct N (P,R350), which has a major absorption maximum between 550 and 560 nm but has lower absorbance than bR570. N was barely detectable at low pH, low ionic strength, and physiological temperature. However, when the thermal relaxation of N to bR570 was inhibited by increasing pH, increasing ionic strength, and decreasing temperature, its relaxation time could be as long as 10 s at room temperature. N is also photoactive; when it is present in significant concentrations, e.g., accumulated by background light, the flash-induced absorption changes of purple membrane suspensions were affected. Double-excitation experiments showed an M-like photoproduct of N,NM, with an absorption maximum near 410 nm and a much longer lifetime than M412. It may be in equilibrium with an L-like precursor NL. We suggest that N occurs after M412 in the photoreaction cycle and that its photoproduct NM decays into bR570. Thus, at high pH and high light intensity, the overall photoreaction of bR may be approximated by the two-photon cycle bR570----M412----N----(NL----NM)----bR570, whereas at neutral pH and low light intensity it can be described by the one-photon cycle bR570----M412----N----O640----bR570.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circular dichroism study of membrane dynamics. Effects of carboxymethyl-chitin

The circular dichroism (CD) active phospholipid bis(4'-n-octanoxyazobenzene-4-carboxyl)-L-alpha-phosphatidylcholin e (CDPC) was used to study the effects of carboxymethyl-chitin (CM-chitin) on the membrane dynamics. For this purpose, CD and electronic spectra were observed for a mixture of CM-chitin and liposomes composed of CDPC and egg lecithin (EPC) (50% CDPC-EPC-liposomes), and for a mixture of CM-chitin, 50% CDPC-EPC-liposomes, and EPC-liposomes. CM-chitin at concentrations higher than ca. 10(-5) M induced the increase of absorbance at 600 nm synchronized with the dilution of CDPC in 50% CDPC-EPC-liposomes by EPC matrix, indicating that CM-chitin induces the fusion among a few liposomes and/or the lipid transfer through the transient liposome fusion. Temperature dependent CD spectra of a mixture of the 50% CDCP-EPC-liposomes and CM-chitin suggested that even high concentration of CM-chitin has no significant perturbation of the lipid organization in the membranes. The effects of CM-chitin on the leakage of [3H]sucrose from the EPC-liposomes were also studied. By the presence of 5 X 10(-6) M CM-chitin, the half-life for leakage of [3H]sucrose in plasma was increased by 4 times. This effect of CM-chitin was reduced by chitinase, while no effect was observed with lysozyme, suggesting that CM-chitin molecules are adsorbed on the liposome surface and stabilize the liposomes against the plasma proteins.     

Isolation and low-temperature preservation of liposomes containing rifampicin

The optimal conditions for preparations of rifampicin-containing liposomes were determined with the methods of mechanical shaking, gas dispersion and and reversible phases. It was found that the percentage of rifampicin incorporation into liposomes depended on the molar ratio of the antibiotic to the lipid (the optimal ratio was 1 : 10), the size and structure of liposomes, the amount of cholesterol added and the lipid membrane charge. Incorporation of rifampicin amounted to 16.1 +/- 2.4, 39.2 +/- 3.2 and 60.5 +/- 2.9 per cent with respect to neutral lecithin multilamellar liposes, liposomes prepared with the gas dispersion method and liposomes prepared with the method of reversible phases, respectively. Cholesterol in a molar ratio to lecithin equal to 2 : 5 or higher and dicetyl phosphate imparting the negative charge to the membrane had an inhibitory effect on the drug uptake by liposomes, while stearyl amine having the positive charge had a stimulating effect. The effect of the cryoprotectors glucose, polyvinylpyrrolidone, poly-ethylene glycole-400 and glycerol on low-temperature preservation and storage of rifampicin-containing liposomes was studied. It was shown that 10--15 per cent solutions of sucrose and glucose had the highest cryoprotective effect, when the two-stage method of freezing was used. It provided almost 84 per cent preservation of liposomal rifampicin. Electron microscopy showed that after defrosting liposomes no significant changes in the size and structure of lipid membranes were observed.     

Nonsymmetrical polymethine dyes as fluorescent probes for the study of microviscosity of membrane phospholipid bilayers

Nonsymmetrical polymethine dyes are shown to be applied as a new approach in the studies of phospholipid membrane microviscosity. The method requires determination of the intensity ratio for the long-wave length (Ig) and short-wave length (Ik) bands of fluorescence spectra in the region of 730-770 nm at exitation 600 nm. It allows determination of microviscosity by comparative measurements of the fluorescence parameter Ig/Ik in the model medium of the known viscosity (glycerol) and the object under study. Microviscosity in egg phosphatidylcholine vesicules (liposomes) is found to be 0.6-1.2 P. It depends on the surface curvature (size of vesicle), cholesterol content and temperature. It the studies of dimiristoylphosphatidyl choline liposomes the changes in microviscosity at the phase transition temperature are shown to be from 4.5 to 1.1 P. The transition temperature is 24.5 degrees C, the transition range being 2.2 degrees C. The results of this work demonstrate the advantages of the suggested approach to study mobility in phospholipid membranes and confirm it to be promising to study natural membranes and whole cells.     

Optical probe responses on sarcoplasmic reticulum. Merocyanine and oxonol dyes.

The fluorescence and absorbance of merocyanine 540 in suspensions of skeletal muscle microsomes is altered by the binding of Ca2+ and other cations to the membrane. The order of effectiveness of various cations in causing this effect is La greater than Ca congruent to Mg greater than K. Competition between Ca2+, Mg2+, and K+ suggests the involvement of low affinity, relatively nonspecific cation binding sites in the process. Changes in the fluorescence and absorbance of merocyanine were also observed during ATP-dependent accumulation of calcium into sarcoplasmic reticulum. These changes are satisfactorily explained by the binding of accumulated calcium to binding sites on the interior of sarcoplasmic reticulum membrane. The small absorbance response of the oxonol dye bis[1,3-dibutylbarbituric acid-(5)]trimethinoxonol to Ca2+ and ATP is qualitatively similar to that of merocyanine 540 and can be readily explained by the same mechanism. We have found no clear evidence that any of the observed dye responses are due to changes in the diffusion potential across the sarcoplasmic reticulum membrane generated by an electrogenic transport mechanism. The possibility is considered that merocyanine and oxonol dyes respond to changes in electrostatic surface potential caused by the binding of cations.     

Involvement of spectrin in the maintenance of phase-state asymmetry in the erythrocyte membrane

The fluorescent probe merocyanine 540 does not stain the plasma membrane of normal human or murine erythrocytes, nor of genetically abnormal human spherocytic erythrocytes. It does, however, stain erythrocyte membranes in several systems in which the underlying spectrin network is altered or missing. Because of the greater affinity of merocyanine 540 for fluid—phase lipid bilayers, these results suggest that the external leaflet of erythrocyte membranes becomes more disordered upon alteration or loss of the internal spectrin network. Analysis of the transbilayer arrangement of membrane phospholipids by digestion with phospholipase A2 suggests that lipid compositional asymmetry of the erythrocyte membrane is responsible for a phase-state asymmetry between the two lipid leaflets, and that spectrin is required to maintain this asymmetry and the gel-like state of the external leaflet.     

Acquired thermotolerance,membrane lipids and osmolytes profiles of xerohalophilic fungus Aspergillus penicillioides under heat shock

Xerophilic fungi accumulate a large amount of glycerol in the cytosol to counterbalance the external osmotic pressure. But during heat shock (HS) majority of fungi accumulate a thermoprotective osmolyte trehalose. Since glycerol and trehalose are synthesized in the cell from the same precursor (glucose), we hypothesised that, under heat shock conditions, xerophiles growing in media with high concentrations of glycerol may acquire greater thermotolerance than those grown in media with high concentrations of NaCl. Therefore, the composition of membrane lipids and osmolytes of the fungus Aspergillus penicillioides, growing in 2 different media under HS conditions was studied and the acquired thermotolerance was assessed. It was found that in the salt-containing medium an increase in the proportion of phosphatidic acids against a decrease in the proportion of phosphatidylethanolamines is observed in the composition of membrane lipids, and the level of glycerol in the cytosol decreases 6-fold, while in the medium with glycerol, changes in the composition of membrane lipids are insignificant and the level of glycerol is reduced by no more than 30%. In the mycelium trehalose level have increased in both media, but did not exceed 1% of dry weight. However, after exposure to HS the fungus acquires greater thermotolerance in the medium with glycerol than in the medium with salt. The data obtained indicate the interrelation between changes in the composition of osmolytes and membrane lipids in the adaptive response to HS, as well as the synergistic effect of glycerol and trehalose.     

Direct observation of singlet oxygen production by merocyanine 540 associated with phosphatidylcholine liposomes

The production of singlet molecular oxygen (1O2) by the photosensitizing dye merocyanine 540 (MC540) bound to phosphatidylcholine liposomes has been demonstrated by direct detection of 1O2 luminescence at 1268 nm. 1O2 phosphorescence emission was enhanced in deuterated buffer and upon saturation of the sample with oxygen and could be quenched by the addition of sodium azide to the external medium. No 1O2 luminescence was detected in nitrogen-saturated samples, in the absence of dye, or with MC540 in aqueous solution. Photobleaching of liposome-bound MC540 was also observed to be dependent on oxygen concentration. These studies are consistent with 1O2 intermediacy in the mechanism of MC540-mediated photosensitization.