The preservation of liposomes by raffinose family oligosaccharides during drying is mediated by effects on fusion and lipid phase transitions

Raffinose family oligosaccharides (RFO) have been implicated as protective agents in the cellular dehydration tolerance, especially of many plant seeds. However, their efficacy in stabilizing membranes during dehydration has never been systematically investigated. We have analyzed the effects of sucrose, raffinose, stachyose, and verbascose on liposome stability during air-drying. With increasing degree of polymerization (DP), the RFO were progressively better able to stabilize liposomes against leakage of aqueous content and against membrane fusion after rehydration. Indeed, there was a very tight linear correlation between fusion and leakage for all RFO. These data indicate that increased protection of liposomes against leakage with increasing DP is due to better protection against fusion. This is in accord with the higher glass transition temperature of the longer chain oligosaccharides. Further evidence for the influence of glass transitions on membrane stability in the dry state was provided by experiments testing the temperature dependence of membrane fusion. During incubation at temperatures up to 95 °C for 2 h, fusion increased less with temperature in the presence of higher DP sugars. This indicates that RFO with a higher glass transition temperature are better able to protect dry membranes at elevated temperatures. In addition, Fourier-transform infrared (FTIR) spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry liposomes in the presence of all investigated sugars. However, the RFO became slightly less effective with increasing chain length, again pointing to a decisive role for preventing fusion. A direct interaction of the RFO with the lipids was indicated by a strong effect of the sugars on the phosphate asymmetric stretch region of the infrared spectrum.     

The preservation of liposomes by raffinose family oligosaccharides during drying is mediated by effects on fusion and lipid phase transitions

Raffinose family oligosaccharides (RFO) have been implicated as protective agents in the cellular dehydration tolerance, especially of many plant seeds. However, their efficacy in stabilizing membranes during dehydration has never been systematically investigated. We have analyzed the effects of sucrose, raffinose, stachyose, and verbascose on liposome stability during air-drying. With increasing degree of polymerization (DP), the RFO were progressively better able to stabilize liposomes against leakage of aqueous content and against membrane fusion after rehydration. Indeed, there was a very tight linear correlation between fusion and leakage for all RFO. These data indicate that increased protection of liposomes against leakage with increasing DP is due to better protection against fusion. This is in accord with the higher glass transition temperature of the longer chain oligosaccharides. Further evidence for the influence of glass transitions on membrane stability in the dry state was provided by experiments testing the temperature dependence of membrane fusion. During incubation at temperatures up to 95 degrees C for 2 h, fusion increased less with temperature in the presence of higher DP sugars. This indicates that RFO with a higher glass transition temperature are better able to protect dry membranes at elevated temperatures. In addition, Fourier-transform infrared (FTIR) spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry liposomes in the presence of all investigated sugars. However, the RFO became slightly less effective with increasing chain length, again pointing to a decisive role for preventing fusion. A direct interaction of the RFO with the lipids was indicated by a strong effect of the sugars on the phosphate asymmetric stretch region of the infrared spectrum.     

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.     

A comprehensive evaluation of the effects and mechanisms of antifreeze proteins during low-temperature preservation

During the past 10 years, it has become clear that the effects of antifreeze proteins (AFPs) on cell viability and on thermodynamic properties during low-temperature preservation are complex, even controversial. In this paper, these studies are reviewed systematically and some conclusions are drawn. It is shown that AFPs can display both protective and cytotoxic actions and both nucleation of ice and inhibition of ice crystal growth, depending on several factors; these include the specific storage protocol, the dose and type of AFP, the composition and concentration of cryoprotectant, and the features of the biological material. A novel model, incorporating some recent findings concerning these proteins, is proposed to explain this dual effect of AFPs during cryopreservation. AFP-ice complexes have some affinity interactions with cell membranes and with many other molecules present in cryopreservation solutions. When the intensity of these interactions reaches a certain level, the AFP-ice complexes may be induced to aggregate, thereby inducing ice nucleation and loss of the ability to inhibit recrystallization.     

Specific interaction of lectins with liposomes and monolayers bearing neoglycolipids

The interaction of three lectins (wheat germ, Ulex europaeus I, and Lotus tetragonolobus agglutinins: WGA, UEA-I and LTA) with either N-acetyl-D-glucosamine or L-fucose neoglycolipids incorporated into phospholipid monolayers and liposome bilayers was studied at the air/water interface and in bulk solution.The results show that for both systems studied, synthesized neoglycolipids were capable of binding their specific lectin and that, in general, the binding of lectins increased with the increase in the molar fraction of the saccharide derivative incorporated in either the monolayers or bilayers. However, whereas for UEA-I, molecular recognition was enhanced by a strong hydrophobic interaction, for WGA and LTA successful recognition was predominantly related to the distance between neighboring sugar groups. The observed lengthy adsorption times of these lectins onto their specific ligands were attributed to interfacial conformational changes occurring in the proteins upon their adsorption at the interfaces.     

Alternative drying processes for the industrial preservation of lactic acid starter cultures

The preservation of lactic acid starter cultures by alternative drying processes has attracted increasing attention due to the high costs and energy consumption of freezing and freeze drying. This review thus aims to provide a survey regarding the state of knowledge of starter culture production at high levels of viability. The results from numerous studies on various drying processes and lactic acid bacteria are summarized. The alternative drying processes considered, such as spray drying, fluidized bed drying, and vacuum drying, are mainly of industrial interest. The features, advantages, and disadvantages of these drying processes are described. In conclusion, the important factors that need to be considered, standardized, or optimized to achieve high levels of viability include intrinsic tolerance of cultures, growth media and conditions, stress induction, cell harvesting conditions, protective agents, rehydration conditions, enumeration of cells, and storage conditions.     

Quantifying the effects of melittin on liposomes

Melittin, the soluble peptide of bee venom, has been demonstrated to induce lysis of phospholipid liposomes. We have investigated the dependence of the lytic activity of melittin on lipid composition. The lysis of liposomes, measured by following their mass and dimensions when immobilised on a solid substrate, was close to zero when the negatively charged lipids phosphatidyl glycerol or phosphatidyl serine were used as the phospholipid component of the liposome. Whilst there was significant binding of melittin to the liposomes, there was little net change in their diameter with melittin binding reversed upon salt injection. For the zwitterionic phosphatidyl choline the lytic ability of melittin is dependent on the degree of acyl chain unsaturation, with melittin able to induce lysis of liposomes in the liquid crystalline state, whilst those in the gel state showed strong resistance to lysis. By directly measuring the dimensions and mass changes of liposomes on exposure to melittin using Dual Polarisation Interferometry, rather than following the florescence of entrapped dyes we attained further information about the initial stages of melittin binding to liposomes.     

Quantifying the effects of melittin on liposomes

Melittin, the soluble peptide of bee venom, has been demonstrated to induce lysis of phospholipid liposomes. We have investigated the dependence of the lytic activity of melittin on lipid composition. The lysis of liposomes, measured by following their mass and dimensions when immobilised on a solid substrate, was close to zero when the negatively charged lipids phosphatidyl glycerol or phosphatidyl serine were used as the phospholipid component of the liposome. Whilst there was significant binding of melittin to the liposomes, there was little net change in their diameter with melittin binding reversed upon salt injection. For the zwitterionic phosphatidyl choline the lytic ability of melittin is dependent on the degree of acyl chain unsaturation, with melittin able to induce lysis of liposomes in the liquid crystalline state, whilst those in the gel state showed strong resistance to lysis. By directly measuring the dimensions and mass changes of liposomes on exposure to melittin using Dual Polarisation Interferometry, rather than following the florescence of entrapped dyes we attained further information about the initial stages of melittin binding to liposomes.     

Proceedings: A method for preservation of bacteria and bacteriophages by drying in vacuo

An efficient and practical method was established to preserve bacterial strains and bacteriophages. The method is characterized by drying without freezing and by use of a cotton wool plug (nonabsorbent) to prevent contamination. Drying conditions were examined by measuring temperature, vacuum, and residual moisture of the samples. From the measurement, it was found that the cotton wool plug acts as a buffer and a desiccant. Thus, the specimens reached optimal conditions during storage. Another point of advantage is that the temperature of the specimen during the drying procedure was 2–5 °C; therefore, the evaporation of the water is rapid and the time of completion is shorter than that during lyophilization.     

Fructans from oat and rye: Composition and effects on membrane stability during drying

Fructans have been implicated in the abiotic stress tolerance of many plant species, including grasses and cereals. To elucidate the possibility that cereal fructans may stabilize cellular membranes during dehydration, we used liposomes as a model system and isolated fructans from oat (Avena sativa) and rye (Secale cereale). Fructans were fractionated by preparative size exclusion chromatography into five defined size classes (degree of polymerization (DP) 3 to 7) and two size classes containing high DP fructans (DP > 7 short and long). They were characterized by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The effects of the fructans on liposome stability during drying and rehydration were assessed as the ability of the sugars to prevent leakage of a soluble marker from liposomes and liposome fusion. Both species contain highly complex mixtures of fructans, with a DP up to 17. The two DP > 7 fractions from both species were unable to protect liposomes, while the fractions containing smaller fructans were protective to different degrees. Protection showed an optimum at DP 4 and the DP 3, 4, and 5 fractions from oat were more protective than all other fractions from both species. In addition, we found evidence for synergistic effects in membrane stabilization in mixtures of low DP with DP > 7 fructans. The data indicate that cereal fructans have the ability to stabilize membranes under stress conditions and that there are size and species dependent differences between the fructans. In addition, mixtures of fructans, as they occur in living cells may have protective properties that differ significantly from those of the purified fractions.     

Fructans from oat and rye: composition and effects on membrane stability during drying

Fructans have been implicated in the abiotic stress tolerance of many plant species, including grasses and cereals. To elucidate the possibility that cereal fructans may stabilize cellular membranes during dehydration, we used liposomes as a model system and isolated fructans from oat (Avena sativa) and rye (Secale cereale). Fructans were fractionated by preparative size exclusion chromatography into five defined size classes (degree of polymerization (DP) 3 to 7) and two size classes containing high DP fructans (DP>7 short and long). They were characterized by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The effects of the fructans on liposome stability during drying and rehydration were assessed as the ability of the sugars to prevent leakage of a soluble marker from liposomes and liposome fusion. Both species contain highly complex mixtures of fructans, with a DP up to 17. The two DP>7 fractions from both species were unable to protect liposomes, while the fractions containing smaller fructans were protective to different degrees. Protection showed an optimum at DP 4 and the DP 3, 4, and 5 fractions from oat were more protective than all other fractions from both species. In addition, we found evidence for synergistic effects in membrane stabilization in mixtures of low DP with DP>7 fructans. The data indicate that cereal fructans have the ability to stabilize membranes under stress conditions and that there are size and species dependent differences between the fructans. In addition, mixtures of fructans, as they occur in living cells may have protective properties that differ significantly from those of the purified fractions.     

Surface effects in preparation of cell-size liposomes

Effects of surface type and area were shown to be important in the yield of cell-size liposomes, but not in determining their size. The liposomes were prepared by dissolving lipids in a chloroform-methanol solution and then evaporating the solvent under nitrogen in the presence of glass beads. After evaporation of the solvent, which was rapid due to the increased surface area, the dried lipids were then swollen in water at high temperatures (higher than the phase transition of the lipids), which led to formation of giant liposomes. The number of liposomes prepared in the presence of pyrex glass beads, which increase more than 100-times the surface area of lipid-glass contact, is more than 5-times larger than in the control experiments without glass beads. The yield of liposomes in the presence of another type of glass bead was almost the same as in the control experiments. These effects may be due to long- and short-range intermolecular interactions in the glass/water/lipid system.     

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.     

Specific suppression of anti-hapten reaginic antibody titers with hapten-coated liposomes

Reaginic antibodies to DNP and ovalbumin (OA) were induced in B6D2F1 mice by a single i.p. injection of 1 microgram of DNP3-OA suspended with 1 mg of A1(OH)3 in 0.5 ml of saline. The anti-DNP reaginic antibody titers were markedly depressed by treatment of mice with DNP-coated liposomes. This treatment, however, did not affect the level of antibody formation to OA.     

Specific binding of mitochondrial protein precursors to liposomes containing cardiolipin

In vitro synthesized precursors of several mitochondrial proteins, including P-450(SCC), adrenodoxin, and malate dehydrogenase, bound to liposomes prepared from mitochondrial phospholipids, but not to those from microsomal phospholipids. When liposomes were prepared from various pure phospholipids, adrenodoxin precursor was bound only to the liposomes that contained cardiolipin. The liposomes containing other phospholipids did not show the binding affinity for the precursor. The binding was observed only with the precursor peptides of adrenodoxin and malate dehydrogenase, and their mature forms were not bound to the liposomes. The binding of the precursors was dependent on the concentration of cardiolipin in the liposomes. Liposomes containing various cardiolipin derivatives with modified polar head groups showed very different binding affinity for adrenodoxin precursor, suggesting the importance of the structure of the polar head of the cardiolipin molecule. Two or three positively charged amino acid residues in the extension peptide of P-450(SCC) precursor were replaced by neutral amino acid residues by site-directed mutagenesis. The mutated P-450(SCC) precursors did not bind to the liposomes containing cardiolipin. The results indicated that mitochondrial protein precursors have specific affinity for cardiolipin, and the affinity was due to the interaction between the extension peptides of the precursors and the polar head of the cardiolipin molecule.     

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.