Spin label and microcalorimetric studies of the interaction of DNA with unilamellar phosphatidylcholine liposomes

High-molecular DNA from chicken erythrocytes interacts with 1,2-dipalmitoylphosphatidylcholine in unilamellar liposomes, both in the presence and absence of Mg2+ ions. This interaction results in a phase separation in liposome membranes. The new phase induced by DNA and Mg2+ has a higher gel-liquid crystal phase transition temperature as measured by microcalorimetry. In the liquid crystalline state, the 16- and 5-doxyl stearic acid spin labels indicate changed local bilayer properties at the label position in the new phase.     

Interaction of clathrin coat proteins with unilamellar and multilamellar vesicles of phosphatidylcholine

The binding of clathrin and accessory coat proteins to small unilamellar vesicles and to liposomes of uncharged phospholipids has been followed by chromatography, 31P-NMR, ESR and fluorescence anisotropy. At pH 6.5 and at an ionic strength value (0.1 M Mes) close to that used during the purification of clathrin-coated vesicles, the proteins do not restore the characteristic network found around the natural vesicles. Instead, a limited fusion leads to enlarged structures in which the perturbation of the dynamics of the phospholipids decreases gradually with the depth in the membrane. While the rate of motion of the outer polar heads is lowered, the order parameter of doxyl groups located either under or in the vicinity of the glycerol backbone is not affected by the proteins. In the inner core of the membrane, the main thermotropic transition of the hydrocarbon chains is unchanged. All the effects are the results of interactions limited to the membrane surface. The electrostatic nature of these interactions is evidenced when the embedded spin labels have a charge protruding at the membrane surface. An 'anchoring' effect appears which is due to the charged groups of the proteins. The lateral diffusion of the probes is reduced and, at low ionic strength, a cationic derivative no longer detects the thermotropic transition of the hydrocarbon chains. These results indicate that, although it is known that clathrin and accessory proteins bind to membranes by a series of protein-protein interactions, this system is not devoid of lipid-protein interactions, at least when it is not organized as in the natural system.     

Kinetic studies on the interaction of phosphatidylcholine liposomes with Triton X-100

Sonicated unilamellar and large multilamellar liposome suspensions have been treated with the non-ionic detergent Triton X-100, and the subsequent changes in turbidity have been studied as a function of time. Sonicated liposome suspensions exhibit an increase in turbidity that takes place in two stages, a fast, low-amplitude one is completed in less than 100 ms, and a slow large-amplitude one occurs in 20-40 s. The first increase in turbidity is associated to detergent incorporation into the bilayer, and the second one, to vesicle fusion. The fast stage may be detected at all detergent concentrations, while the slow one is only seen above the critical micellar concentration of Triton X-100. Both processes may be interpreted in terms of first-order kinetics. Studies of the variation of kexp with lipid and detergent concentration suggest a complex multi-step mechanism. In the case of multilamellar liposomes, a fast increase in turbidity is also seen after detergent addition, which is followed by a slow (20-60 s) decrease in turbidity and a very slow (up to 12 h) large scale decrease in turbidity. These processes do not conform to single-exponential patterns. The fast stage is also thought to reflect surfactant incorporation, while the decrease in turbidity is interpreted as bilayer solubilization starting with the outer bilayer (slow stage) and proceeding through the remaining ones (very slow stage).     

Topological distribution of bovine serum albumin in multilamellar and unilamellar liposomes

The topological distribution of bovine serum albumin (BSA) in multilamellar vesicles (MLV) and unilamellar vesicles (ULV) composed of 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DML)/cholesterol (molar ratio, 3:1) was studied by ESR using hydrophobic spin-labelled lecithins and hydrophilic Tempocholine. A spin-labelled BSA was also prepared, characterized and used as a probe. Results with hydrophobic spin-labelled lecithin probes showed no significant phospholipid-albumin interaction, indicating the virtual absence of albumin from the phospholipid bilayer of MLV and ULV. Reduction with L-ascorbic acid showed that MLV contained about 50% and ULV 90% of spin-labelled albumin on the surface. The distribution of Tempocholine in MLV and ULV was similar to that of entrapped BSA. These findings were confirmed by results using liposomes treated with nickel which broadened the ESR spectra of probes on the surface of vesicles. This study and our previous work suggest that the immunoadjuvant effect of liposomes can be mediated by surface antigens and can be maximized by preferential surface distribution as in ULV-associated BSA.     

Partition of piperidinoethylesters of 2-alkyloxyphenylcarbamic acid in unilamellar phosphatidylcholine liposomes.

Molar partition coefficients for amphiphilic N-[2-(2-alkyl-oxyphenyl-carbamoyloxy)-ethyl]-piperidinium chlorides (PAA) between small unilamellar egg yolk phosphatidyl choline liposomes and saline, as determined by ultraviolet difference spectroscopy at 22 degrees C, pH 5-6, v = 34640 cm-1, and at 100 mumol/l PAA concentration, were 149, 1990, and 7474 for PAA with 5, 7, and 9 carbon atoms in the alkyloxy substituent, respectively. At the PAA concentration used, the cut-off in biological activities of PAAs with long alkyloxy substituents could not be caused by the self-association of PAA molecules in the aqueous phase.     

The interaction of adriamycin with small unilamellar vesicle liposomes. A fluorescence study

The interaction of the antineoplastic agent adriamycin with sonicated liposomes composed of phosphatidylcholine alone and with small amounts (1-6%) of cardiolipin has been studied by fluorescence techniques. Equilibrium binding data show that the presence of cardiolipin increases the amount of drug bound to liposomes when the bilayer is below its phase transition temperature and when the ionic strength is relatively low (0.01 M). At higher ionic strength (0.15 M) and above the Tm (i.e. conditions which are closer to the physiological state) the binding of the drug to the two liposome types is nearly the same. Thus the differences in the interactions of adriamycin with cardiolipin-containing membranes, as opposed to those composed of phosphatidylcholine alone, are not due simply to increased binding but rather to an altered membrane structure when this lipid is present. Quenching of adriamycin fluorescence by iodide shows that bound drug is partially, but not completely, buried in the liposomal membrane. Both in the presence and absence of cardiolipin the bulk of the adriamycin is more accessible to the quencher below the Tm than above it; that is, a solid membrane tends to exclude the drug from deep penetration. Above the Tm, the presence of cardiolipin alters the nature of liposome-adriamycin interaction. Here the fluorescence quenching data suggest that the presence of small amounts of cardiolipin (3%) in a phosphatidylcholine matrix creates two types of binding environments for drug, one relatively exposed and the other more deeply buried in the membrane. The temperature dependence of the adriamycin fluorescence and the liposome light scattering reveal that cardiolipin alters the thermal properties of the bilayer as well as its interaction with adriamycin. At low ionic strength lateral phase separations may occur with both pure phosphatidylcholine and when 3% cardiolipin is present; under these conditions the bound adriamycin exists in two kinds of environment. It is notable that only adriamycin fluorescence reveals this phenomenon; thebulk property of liposome light scattering reports only on the overall membrane phase change. These data suggest that under certain conditions the drug binding sites in the membranes are decoupled from the bulk of the lipid bilayer.     

Use of phospholipid exchange protein to measure inside-outside transposition in phosphatidylcholine liposomes

The exchange of phosphatidylcholine between [³²P]phosphatidylcholine liposomes and unlabeled mitochondria was catalyzed by a purified phospholipid exchange protein from bovine heart cytosol. The loss of [³²P]phosphatidylcholine from the liposomes appeared to proceed in two stages: with 100 units of phospholipid exchange protein per ml the half-time of initial stage was about 10 min and that of the final stage 4 days or greater. Agarose-gel chromatography of the liposomes showed an elution compatible with a homogeneous pool of small single walled vesicles. Treatment of phosphatidyl [¹⁴C]choline liposomes with phospholipase D (phosphatidylcholine phosphatidohydrolase) showed that labeled phospholipid removable during the rapid exchange phase was subject to hydrolysis by the phospholipase, but that the labeled phospholipid left after the rapid exchange was completed could not be hydrolyzed by phospholipase D. It is proposed that the rapidly exchanging phosphatidylcholine constitutes the outer layer of the liposome bilayer. The long half-lives of 4 days or more probably represent the transposition of Phosphatidylcholine from the inner to the outer layer of the liposome bilayer.     

The interaction of Bacillus protoplasts with sonicated phosphatidylcholine liposomes

When protoplasts from Bacillus subtilis are incubated with sonicated liposomes made from egg-yolk phosphatidylcholine, this phospholipid is incorporated into the protoplast membranes. Biochemical, fluorescence and ultrastructural data suggest that incorporation occurs through membrane fusion.     

1H- and 2H-n.m.r. studies of water in gamma-irradiated phosphatidylcholine multilamellar liposomes

1H- and 2H-n.m.r. studies of gamma-irradiation-induced variations in the dynamic structure and proportional amounts of free, trapped and bound water species in multilamellar liposomes are reported and discussed. Bound water is shown to increase with dose and to be present in two different structural states. A dose-dependent decrease in the 1H-n.m.r. relaxation times of bound water following gamma-irradiation is reported. Variations are suggested as being due to large scale changes at the bilayer surface.     

Determination of the hydrophobic binding site of phosphatidylcholine exchange protein with photosensitive phosphatidylcholine.

1-Acyl-2-(7-(4-azido-2-nitrophenoxy)-[1-14C]heptanoly)-sn-glycero-3-phosphocholine was synthesized in order to study the lipid-binding site of the phosphatidylcholine exchange protein from bovine liver. Photosensitive phosphatidylcholine was incorporated into the protein by incubation with vesicles of this phosphatidylcholine derivative. The lipid-protein complex was separated from the vesicles by chromatography on Biogel A-0.5m. Photolysis of the complex by irradiation with light of a high pressure mercury lamp at a wavelength above 340 nm generated the highly reactive nitrene. Sodium dodecyl sulfate gel electrophoresis of the photolysed complex indicated that 30% of the endogenous 14C-labeled phosphatidylcholine was covalently linked to the protein. Peptides were isolated after digestion of the photolysed complex with protease from Staphylococcus aureus and trypsin. It was determined that the 2-acyl chain of the phosphatidylcholine molecule was linked to the peptide segment -Gly-Ser-Lys-Val-Phe-Met-Tyr-Tyr-. This segment was part of a protease peptide of about 65 residues of which the sequence was determined by Edman degradation for the first 38 residues. This peptide contains a cluster of apolar residues -Val-Phe-Met-Tyr-Tyr-Phe with an extremely high hydrophobicity index and with a predicted beta-sheet conformation. It is concluded that this hydrophobic cluster forms part of the binding site.     

Generation of multilamellar and unilamellar phospholipid vesicles

Multilamellar and unilamellar vesicles can be generated by a variety of techniques which lead to systems with differing lamellarity, size, trapped volume and solute distribution. The straight-forward hydration of lipid to produce multilamellar vesicles (MLVs) results in systems which exhibit low trapped volumes and where solutes contained in the aqueous buffer are partially excluded from the MLV interior. Large trapped volumes and equilibrium solute distributions can be achieved by freeze-thawing or by ‘reverse phase’ procedures where the lipid is hydrated after being solubilized in organic solvent. Unilamellar vesicles can be produced directly from MLVs by extrusion or sonication or, alternatively, can be obtained by reverse phase or detergent removal procedures. The advantages and limitations of these techniques are discussed.     

Fluorochrome staining of multilamellar liposomes.

Multilamellar liposomes can be stained with such fluorochromes as acridine orange, eosin Y, neutral red, and thiazine red. The liposomes are brought into a 1% solution of the fluorochrome; 5-10 minutes later they are centrifuged and washed by resuspending in water or phosphate buffered saline three times. The last pellet is resuspended and a drop studied with the fluorescence microscope (1000 x magnification). The fluorochrome is seen to be accumulated in the liposomal membranes. Acridine orange could also be trapped in the aqueous compartments of the liposomes but the trapped fluorochrome was gradually lost from the liposomes. Part of the fluorochrome, however, remained associated with the liposomal membranes for a long time. Additional experiments justify the conclusion that an equilibrium is maintained between fluorochromes in the aqueous and lipid phases.     

The effects of charge and size on the interaction of unilamellar liposomes with macrophages

The effect of the positive surface charge of unilamellar liposomes on the kinetics of their interaction with rat peritoneal macrophages was investigated using three sizes of liposomes: small unilamellar vesicles (approx. 25 nm diameter), prepared by sonication, and large unilamellar vesicles (100 nm and 160 nm diameter), prepared by the Lipoprep dialysis method. Charge was varied by changing the proportion of stearylamine added to the liposomal lipids (egg phosphatidylcholine and cholesterol, molar ratio 10:2.5). Increasing the stearylamine content of large unilamellar vesicles over a range of 0-25 mol% enhanced the initial rate of vesicle-cell interaction from 0.1 to 1.4 microgram lipid/min per 10(6) cells, and the maximal association from 5 to 110 micrograms lipid/10(6) cells. Cell viability was greater than 90% for cells incubated with large liposomes containing up to 15 mol% stearylamine but decreased to less than 50% at stearylamine proportions greater than 20 mol%. Similar results were obtained with small unilamellar vesicles except that the initial rate of interaction and the maximal association were less sensitive to stearylamine content. The initial rate of interaction, with increasing stearylamine up to 25 mol%, ranged from 0.5 to 0.7 microgram lipid/min per 10(6) cells, and the maximal association ranged from 20 to 70 micrograms lipid/10(6) cells. A comparison of the number and entrapped aqueous volume of small and large vesicles containing 15 mol% stearylamine revealed that although the number of large vesicles associated was 100-fold less than the number of small vesicles, the total entrapped aqueous volume introduced into the cells by large vesicles was 10-fold greater. When cytochalasin B, a known inhibitor of phagocytosis, was present in the medium, the cellular association of C8-LUV was reduced approx. 25% but association of SUV increased approx. 10-30%. Modification of small unilamellar vesicles with an amino mannosyl derivative of cholesterol did not increase their cellular interaction over that of the corresponding stearylamine liposomes, indicating that cell binding induced by this glycolipid may be due to the positive charge of the amine group on the sugar moiety. The results demonstrate that the degree of liposome-cell interaction with macrophages can be improved by increasing the degree of positive surface charge using stearylamine. Additionally, the delivery of aqueous drugs to cells can be further improved using large unilamellar vesicles because of their greater internal volume. This sensitivity of macrophages to vesicle charge and size can be used either to increase or reduce liposome uptake significantly by this cell type     

Raman spectroscopic investigation of the interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes.

The interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes is investigated by Laser-Raman spectroscopy. As revealed by the methylene C-H stretching mode the phase transition of the hydrocarbon chains near 40 degree C is eliminated in the presence of gramicidin A. Liposomes prepared from a mixture of lecithin and cholesterol seem to be unaffected by gramicidin A and show only the normal broadened phase transition.     

The effect of stobadine on the copper-induced peroxidation of egg yolk phosphatidylcholine in multilamellar liposomes

Stobadine, (-)- cis-2,8-dimethyl-2 ,3,4,4a,5,9b-hexahydro-1H-pyrido-[4,3b]-indole, is a pyridoindole derivative with antioxidant, antiarrhytmic, neuroprotective, local anesthetic, alpha-adrenolytic, antihistaminic, myorelaxant and other pharmacodynamic effects. The antioxidant properties were tested in a model system of egg yolk phosphatidylcholine (EYPC) multilamellar liposomes. The lipoperoxidation was induced by adding Cu2+ ions and tert-butylhydroperoxide. The course of EYPC peroxidation was monitored spectrophotometrically for conjugated diene formation. We found that stobadine prolonged the lag phase and decreased the rate of peroxidation during the lag phase in a dose-dependent manner. Surprisingly, an increase in the rate of peroxidation was observed at low stobadine concentration in the propagation phase. The possible cause of prooxidative action of stobadine is discussed.     

Fraction of cholesterol undergoing spontaneous exchange between small unilamellar phosphatidylcholine vesicles

The kinetics of the spontaneous exchange of [3H]cholesterol between small unilamellar vesicles of phosphatidylcholine has been reexamined. Although first-order exchange kinetics were observed (k = 0.0117 min-1), in good agreement with previous studies, about 20% of the total cholesterol was found to be nonexchangeable in the 8-h time frame of the experiments. The size of this nonexchangeable pool was found to depend on the type of phospholipid and the temperature. It seems probable that the two pools of cholesterol defined in these experiments reflect the complex phase structure of the cholesterol-phosphatidylcholine vesicles.     

X-ray diffractometry and calorimetry studies of structural modifications induced by gamma-irradiation in phosphatidylcholine multilamellar liposomes

Experimental results are reported on structural and thermodynamic modifications induced by gamma-irradiation in model membranes. Differential scanning calorimetry (DSC) and X-ray diffraction were used to study the different phases and associated transitions of dipalmitoylphosphatidylcholine after 60Co gamma-irradiation. Changes were observed in the shape of calorimetric peaks and in the corresponding enthalpy. The repetition distance of the layers increases while the distances related to the aliphatic chains decrease as a function of gamma-irradiation time. Moreover, an increase in the hexagonal symmetry with increasing dose was detected. No disappearance of a pre-transition was detected even at high doses.     

Raman spectroscopic investigation of the interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes

The interaction of gramicidin A with dipalmitoyl phosphatidylcholine liposomes is investigated by Laser-Raman spectroscopy. As revealed by the methylene CH stretching mode the phase transition of the hydrocarbon chains near 40°C is eliminated in the presence of gramicidin A. Liposomes prepared from a mixture of lecithin and cholesterol seem to be unaffected by gramicidin A and show only the normal broadened phase transition.