Calcium-dependent aggregation and fusion of phosphatidylcholine liposomes induced by complexes of flavonoids with divalent iron

It was found that complexes of the flavonoids quercetin, taxifolin, catechin and morin with divalent iron initiated an increase in light scattering in a suspension of unilamellar 100nm liposomes. The concentration of divalent iron in the suspension was 10μM. Liposomes were prepared from 1-palmitoyl-2-oleoylglycero-3-phoshpatidylcholine. The fluorescent resonance energy transfer (FRET) analysis of liposomes labeled with NBD-PE and lissamine rhodamine B dyes detected a slow lipid exchange in liposomes treated with flavonoid-iron complexes and calcium, while photon correlation spectroscopy and freeze-fracture electron microscopy revealed the aggregation and fusion of liposomes to yield gigantic vesicles. Such processes were not found in liposomes treated with phloretin because this flavonoid is unable to interact with iron. Rutin was also unable to initiate any marked changes because this water-soluble flavonoid cannot interact with the lipid bilayer. The experimental data and computer calculations of lipophilicity (cLogP) as well as the charge distribution on flavonoid-iron complexes indicate that the adhesion of liposomes is provided by an iron link between flavonoid molecules integrated in adjacent bilayers. It is supposed that calcium cations facilitate the aggregation and fusion of liposomes because they interact with the phosphate moieties of lipids.     

An X-ray diffraction and differential scanning calorimetric study on the effect of sucrose on the properties of phosphatidylcholine bilayers

The influence of sucrose, between 0 and 70% in the aqueous phase, upon multilamellar liposomes of dimyristoylphosphatidylcholine was examined by differential scanning calorimetry and X-ray diffraction analysis. Increasing concentrations of sucrose increase the temperatures of both the main transition and the pretransition of the lipid. The effect is greater on the pretransition than on the main transition. At 35 degrees C the interlamellar spacing in the multilamellar liposomes is reduced by increasing sucrose concentration in the aqueous phase and no significant effects are seen in the chain lattice of the bilayers. This result is interpreted as a dehydrating effect of sucrose upon the bilayer-water system at 35 degrees C. At 5 degrees C the interlamellar spacing is increased and this increase is, at high (70%) sucrose concentrations, attributable to an untilting of the lipid acyl chains with no change in the thickness of the aqueous layers in the multilamellae.     

Stabilization of large multilamellar liposomes by human serum in vitro

The leakage of 5,6-carboxyfluorescein from large multilamellar liposomes prepared from dipalmitoylphosphatidylcholine (without or with cholesterol) was investigated in vitro in the presence of human serum. Below the phospholipid phase transition temperature, the rate of dye release is retarted 3–8-fold in the presence of up to 25% human serum in the incubation medium, as compared to the release in isotonic phosphate-buffered saline. This effect is significantly augmented by incorporation of 50 mol% cholesterol into the lipid bilayer. At and above the phase transition temperature, the initial rapid dye leakage in the presence of serum is followed by a slow long-term release. Incubation of the liposomes with serum is assumed to result in the association of serum proteins with the outermost lipid bilayer which in turn will lead to their stabilization, while the inner lamellae are not immediately accessible to the serum proteins. The permeability of the outer protein-rich lipid bilayer appears to be restricted, as concluded from the decreased dye release in the presence of serum. Massive leakage from multilamellar liposomes appears to be primarily due to bilayer defects occurring in the lipid transition region rather than being caused by protein-lipid interactions. The results of our in vitro experiments are discussed in terms of the potential usefulness of multilamellar liposomes as drug carriers in vivo for local and topical applications.     

Thermotropic behavior of liposomes from egg yolk lecithin, hydrogenized egg yolk lecithin and their mixtures

The thermotropic properties of multilamellar liposomes from egg yolk lecithin, hydrogenized egg yolk lecithin and several mixtures of these two lipids were studied with the application of excimer--forming optical probe pyrene and microcalorimetry. It was discovered that when the proportion of the egg yolk lecithin in the lipid mixture was raised the temperature of the main phase transition reduced. For all this, independent of the lipid mixture composition when the temperature was raised, apparently, polarity of pyrene microenvironment in the liposomes bilayers decreased. On the basis of the analysis of solidus and liquidus curves obtained from calorimetric studies of the lipid mixtures and bend points of Arrhenius anamorphose obtained during the pyrene excimer formation measurements some conclusions were made about the role of unmodified and hydrogenized egg yolk lecithin cluster formation in the determination of thermotropic properties of the liposomes from the above two lipids mixtures. High temperature phase transition discovered for the egg yolk lecithin while measuring the pyrene excimer formation is proposed to be closely connected with temperature-dependent changes in the organization of phospholipid heads on the interphase bilayer/H2O solution.     

Effects of phenylamide herbicides on the physical properties of phosphatidylcholine membranes

A number of phenylamide herbicides are observed to uncouple electron transport in isolated chloroplasts and mitochondria and alter the H+ permeability of artificial liposomes. Several of these phenylamides were incorporated into phosphatidylcholine multilamellar and small unilamellar vesicles to measure their effects on the physical properties of membranes. X-ray diffraction analysis of the multilamellar vesicles revealed that the herbicides partitioned into the hydrocarbon chain region of the bilayer, but caused only minimal perturbations on hydrocarbon chain packing. 31P-NMR spectroscopy of these multilamellar vesicles showed both a broadening and lowering of the phase transition temperature of the bilayer lipids upon addition of the herbicides. 13C-NMR spectroscopy of small, unilamellar phosphatidylcholine vesicles was performed to measure the effects of the phenylamides on the chemical shifts and the spin-lattice relaxation times of the individual phosphatidylcholine carbon atoms. None of the added compounds had any measurable effect on the 13C-NMR chemical shifts of the phosphatidylcholine. However, the herbicides significantly modified spin-lattice relaxation times of certain of the lipid carbon atoms. These results generally indicate that the herbicides orient in the lipid bilayers such that the hydrocarbon chains of the phenylamides associate with the hydrocarbon chains of the lipid, whereas the phenyl moiety resides in the polar region of the bilayer.     

Translocation of histone proteins across lipid bilayers and Mycoplasma membranes

We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).     

Thermotropic changes in the conductivity of black bilayers from egg phosphatidylethanolamine

Current fluctuations in black bilayers from phosphatidyl ethanolamine obtained from egg lecithin were registered in the temperature region of the main phase transition of this phospholipid and the bilayer--hexagonal phase transition about 35 degrees; they correspond to the conductivity changes of hundreds of pSm. This transition takes place in the same temperature region as shown by 31P-NMR and depolarization of light-scattering method in phosphatidyl ethanolamine multilamellar liposomes. The scheme of bilayer transformation into hexagonal phase in the temperature region of lipid polymorphic transition is discussed.     

Fluorimetric detection of phospholipid vesicles bound to planar phospholipid membranes.

The first step in the fusion of two phospholipid membranes culminates in the aggregation of the two lipid bilayers. We have used a custom-built fluorimeter to detect multilamellar vesicles (liposomes) containing the fluorescent dye, 6-carboxyfluorescein (6-CF), bound to a planar lipid bilayer (BLM). Liposomes were added to one side of the BLM, and unbound vesicles were perfused out. This left a residual fluorescence from the BLM, but only when the membranes contained anionic lipids, and then only when millimolar levels of calcium were present. This residual fluorescence was consistently detected only when calcium was included in the buffer during the perfusion. This residual fluorescence originated from liposomes bound to the BLM. Breaking the BLM or lysing the adsorbed vesicles with distilled water abolished it. free 6-CF and/or calcium in the absence of liposomes resulted in no residual fluorescence. No residual fluorescence was detected when both the liposomes and the BLM were composed entirely of zwitterionic lipids. This was found to result from the insensitivity of the fluorimeter to a small number of liposomes adsorbed to the BLM. For this system, we conclude that calcium is necessary for both the initiation and maintenance of the state in which the vesicle membrane is bound to the planar bilayer when the membranes contain negatively charged lipids. This attachment is stronger than the interaction between zwitterionic membranes.     

Sublytic and lytic effects of the zwitterionic bile derivative 3-((3-deoxycholamidopropyl)dimethylammonio)-1-propanesulfonate on phosphatidylcholine liposomes

The effects of the zwitterionic bile derivative 3-((3-deoxycholamidopropyl)dimethyl-ammonio)-1-propanesulfonate (Chaps) on multilamellar phosphatidylcholine liposomes have been characterized. When the surfactant is added to preformed liposome suspensions, equilibrium is attained in less than 6 h. Fifty percent solubilization, as measured by analysis of lipid P in supernatants after solubilization, occurs at a 0.32 lipid/detergent mole ratio for a 1 mM phospholipid concentration. Fifty percent release of entrapped glucose occurs at the same detergent concentration, suggesting that, in this system, no increase in permeability occurs prior to solubilization. A linear relationship is found between phospholipid concentration and amount of surfactant producing 50% solubilization. No lytic effect of Chaps is seen below 2 mM surfactant, this being probably near the critical micellar concentration of the amphiphile under our conditions. In the sublytic range of detergent concentrations, Chaps binds the lipid bilayers with high affinity, so that, at least at 1 mM phospholipid, the amount of free Chaps is negligible; solubilization starts when about two surfactant molecules are incorporated per phospholipid molecule. Differential scanning calorimetry shows that incorporation of Chaps into saturated phosphatidylcholine bilayers, even at concentrations below those producing solubilization, causes a decrease in the Tc gel-to-liquid crystalline main transition temperature of the phospholipid, and a decrease in the transition enthalpy; at the same time, a "shoulder" appears on the low-temperature side of the main endotherm. The ensemble of our data suggests that the behavior of Chaps toward phospholipid bilayers is intermediate between that of the natural bile derivatives and that of some well-known nonionic synthetic surfactants.     

Interaction of Adriamycin and N-Trifluoroacetyladriamycin-14-Valerate with Cardiolipin-Containing Lipid Bilayers

Abstract

The interaction of adriamycin (ADR) and N-Trifluoroacetyladriamycin-14-valerate (AD 32) with cardiolipin (CL)-containing multilamellar vesicles was studied by high-sensitivity differential scanning calorimetry, using liposomes formed from either dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) containing small amounts of CL. the drugs partitioned into cardiolipin-containing neutral and acidic bilayers in a manner similar to that observed earlier with CL-free bilayers. the partition coefficient of adriamycin in bilayers of vesicles prepared from DPPG or DPPG in admixture with CL was much higher than that obtained with neutral DPPC vesicles or the DPPC together with CL. Under all conditions, AD 32 was essentially completely partitioned into the lipid phase of the investigated phospholipid membranes. As expected, addition of adriamycin to CL-containing vesicles did not significantly change the thermotropic behavior of these bilayers, whereas the fluidizing effect of AD 32 was directly related to the CL content of the vesicles. the multipeak transitions produced by the anthracyclines in pure DPPG bilayers were preserved in the presence of CL, but the endotherms were broader and slightly shifted to lower temperatures, a finding indicative of stronger interactions. Chlorpromazine (CPZ), which was used as a reference compound to compare the effects produced by the anthracyclines, was found to behave similarly to AD 32 and to be more effective than quinidine (QND), in agreement with their behavior in CL-free liposomes.     

Thermodynamic factors determining the permeability barrier properties of lipid bilayers

The permeability barrier properties of lipid bilayers are usually determined by the rate of swelling of multilamellar liposomes or by the exchange of radioactively labeled molecules in sonicated vesicles. The values reported in the literature for the permeability of water and non electrolytes differ according to which method is applied in their determination. In addition, drastic assumptions (i.e. homogeneity of the membrane) are commonly introduced for the interpretation of the phenomenological permeability coefficients. This paper discusses the permeability coefficient considering the departures from the ideality of the membrane system. The non ideal terms can be put in function of measurable quantities such as the excluded volume of the membrane and the hydration degree of the lipid molecules. By means of this formalism it is possible to explain quantitatively the experimental values found for the permeability coefficient of water in sonicated vesicles below and above the phase transition temperature. In addition, different magnitudes of the energies of activation for the permeation of non electrolytes have been found depending on if the liposomes are dispersed in isotonic or hipertonic solutions of a permeant. The formalism described allows to explain such differences in terms of the influence of the solute concentration on the density of the lipid membrane. The reasons for which the simple formalism for homogeneous membranes can not be applied to lipid membranes are discussed in detail.     

Membrane contact, fusion, and hexagonal (HII) transitions in phosphatidylethanolamine liposomes

The behavior of phosphatidylethanolamine (PE) liposomes has been studied as a function of temperature, pH, ionic strength, lipid concentration, liposome size, and divalent cation concentration by differential scanning calorimetry (DSC), by light scattering, by assays measuring liposomal lipid mixing, contents mixing, and contents leakage, and by a new fluorometric assay for hexagonal (HII) transitions. Liposomes were either small or large unilamellar, or multilamellar. Stable (impermeable, nonaggregating) liposomes of egg PE (EPE) could be formed in isotonic saline (NaCl) only at high pH (greater than 8) or at lower pH in the presence of low ionic strength saline (less than 50 mOsm). Bilayer to hexagonal (HII) phase transitions and gel to liquid-crystalline transitions of centrifuged multilamellar liposomes were both detectable by DSC only at pH 7.4 and below. The HII transition temperature increased, and the transition enthalpy decreased, as the pH was raised above 7.4, and it disappeared above pH 8.3 where PE is sufficiently negatively charged. HII transitions could be detected at high pH following the addition of Ca2+ or Mg2+. No changes in light scattering and no lipid mixing, mixing of contents, or leakage of contents were noted for EPE liposomes under nonaggregating conditions (pH 9.2 and 100 mM Na+ or pH 7.4 and 5 mM Na+) as the temperature was raised through the HII transition region. However, when aggregation of the liposomes was induced by addition of Ca2+ or Mg2+, or by increasing [Na+], it produced sharp increases in light scattering and in leakage of contents and also changes in fluorescent probe behavior in the region of the HII transition temperature (TH). Lipid mixing and contents mixing were also observed below TH under conditions where liposomes were induced to aggregate, but without any appreciable leakage of contents. We conclude that HII transitions do not occur in liposomes under conditions where intermembrane contacts do not take place. Moreover, fusion of PE liposomes at a temperature below TH can be triggered by H+, Na+, Ca2+, or Mg2+ or by centrifugation under conditions that induce membrane contact. There was no evidence for the participation of HII transitions in these fusion events.     

A scanning calorimetric study of the interaction of anthracyclines with neutral and acidic phospholipids alone and in binary mixtures

High sensitivity differential scanning calorimetry was employed to study the thermotropic behavior of multilamellar vesicles of neutral and acidic phospholipids and binary mixtures thereof in the presence of anthracycline antibiotics. Adriamycin and its lipophilic analogue, N-trifluoroacetyladriamycin-14-valerate (AD32) were investigated and compared to chlorpromazine and quinidine with respect to their ability to affect the pretransition and the main transition of the phospholipids suspended in physiological buffer. With liposomes of neutral dipalmitoylphosphatidylcholine the observed effects paralleled to some extent the corresponding octanol/buffer partition coefficients, with adriamycin being the least effective. Calorimetric measurements on liposomes prepared from pure dipalmitoylphosphatidylglycerol or from binary mixtures of dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine showed that modulation of bilayer properties by adriamycin was greatly enhanced in the presence of negatively charged lipid headgroups presumably as a result of electrostatic interactions. AD32 interacted differently from adriamycin with the acidic bilayers at low drug concentrations, in a manner similar to that of its interaction with neutral bilayers. At high drug concentrations both adriamycin and AD32 produced transitions with multiple peaks not exhibited by chlorpromazine and quinidine which may be the result of a specific association of the anthracyclines with dipalmitoylphosphatidylglycerol. All four drugs produced only minor changes in the enthalpy of the main transition of the investigated lipids. The present findings are discussed in terms of their possible physiological relevance.     

Structural effects of neutral lipids on divalent cation-induced interactions of phosphatidylserine-containing bilayers.

Ca2+ is known to induce the adhesion and collapse of phosphatidylserine (PS) bilayers into dehydrated multilamellar structures. The aim of this study was to examine how that interaction and the resultant structures might be modified by neutral lipid species. A combination of rapid mixing, x-ray diffraction, thin-layer chromatography, density gradient centrifugation, and freeze-fracture electron microscopy was used in conjunction with osmotic stress techniques to characterize the structures formed by the Ca(2+)-induced interaction of multilamellar liposomes and of large unilamellar vesicles. The results showed that dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine at concentrations of up to approximately 30 mol % are accommodated in a single dehydrated multilamellar structure. Similar results were obtained using mixed PS species isolated from bovine brain. Principally, the data indicate that neutral lipid is both dehydrated during the rapid collapse process of Ca(PS)2 formation and accommodated within this dehydrated structure. The large energies available on formation of the Ca(PS)2 bilayers contribute to the dehydration of neighboring neutral lipids that likely form continuous bilayers with them. Higher concentrations of these neutral lipids modify Ca(2+)-induced bilayer interactions, leading to progressively weaker interactions, larger bilayer separations, and in some cases separation into two structures; phosphatidylethanolamine species favoring nonbilayer structures tended to promote such separation at lower concentrations than bilayer lipids.     

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.     

pH dependent changes in the reactivity of the primary electron acceptor of system II in spinach chloroplasts to external oxidant and reductant.]

Two pure phospholipids, dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, have been studied using freeze-fracture electron microscopy and the partitioning of the spin label, TEMPO. It is found that the characteristic band pattern, corresponding to monoclinic symmetry in multilamellar liposomes, is observed only in freeze-fracture electron microphotographs when samples are quenched from temperatures intermediate between the chain melting transition temperature and the pretransition temperature of the membrane. Markings are also observed on fracture faces of samples quenched from below the pretransition, but these "bands" are few in number and are widely and irregularly spaced. The lipid membranes used for freeze-fracture were prepared using detergent dialysis and are thought to consist of one, two, or some small number of concentric bilayer shells. These observations are in excellent accord with the recent, prior studies of Janiak, M.J., Small, D.M. and Shirley, G.G., ((1976) Biochemistry 15, 4575--4580), who found monoclinic symmetry (Pbeta' structure) in multilamellar liposomes of these phospholipids only when the sample temperature was intermediate between the main, chain melting transition temperature, and the pretransition temperature. The significance of these results for relating freeze-fracture electron microphotographis to phase diagrams derived from spin label or calorimetric data is discussed briefly. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) partitioning data show distinct differences between liposomal preparations of these lipids, and other preparations having fewer bilayers per vesicular structure, with respect to the position, width, and hysteresis of the pretransition.     

The structural organization of cationic lipid-DNA complexes

The interaction of cationic liposomes with supercoiled plasmid DNA results in a major rearrangement of each component to form compact multilamellar structures comprised of alternating layers of two-dimensional arrays of DNA sandwiched between lipid bilayers. Fluorescence resonance energy transfer was used to estimate the distance of closest approach of DNA to the lipid bilayers in these complexes. The effect of several compositional variables on this distance, including the ratio of cationic lipid to DNA, and the charge density, intrinsic curvature, and fluidity of the lipid bilayer were examined. Additionally, the effect of ionic strength was studied. For complexes prepared at or above a 3:1 charge ratio (+/-), the observed distance of closest approach was found to be in agreement with the intercalation of DNA between lipid bilayers. As the charge ratio was decreased, a monotonic increase in the distance was observed with a maximum observed at 0.5:1. Correlations between differences in the proximity of DNA to the lipid bilayer and the hydrodynamic size of the complexes were also found. A model based on these observations and previous reports suggests the formation of discrete populations of complexes below a charge ratio of 0.5:1 and above 3:1. The structure of the negatively charged complexes is consistent with DNA extending from the surface of the particles, whereas those possessing excess positive charge were multilamellar aggregates with the DNA effectively condensed between lipid bilayers. Complexes between these two states consist of weighted fractions of these two species.     

Permeability of vesicular phospholipid bilayer membranes to thallium and its facilitation

The rate of release of TI⁺ from phospholipid vesicles of different composition was measured by pulse polarography as a function of temperature or in the presence of valinomycin, tetraphenylboron (TPB⁻) or dipicrylamine (DPA⁻) as transport facilitators. The release from pure dipalmitoylphosphatidylcholine (DPPC) vesicles increased abruptly around the pretransition temperature. The release from lipid mixtures with broad transition temperature region increased continuously with temperature. The steepness of the increase decreased with the width of the transition peak. Valinomycin, TPB⁻ (tetraphenylboron) and DPA⁻ (dipicrylamine) facilitate release of TI⁺ from unilamellar vesicles above their phase transition temperature with a first-order release rate constant. They do not facilitate release below the phase transition. Bursts of release were observed upon their addition to the vesicles but after annealing, which was completed within less than a minute, the vesicles were resealed. No facilitated release from multilamellar vesicles could be discerned. The entrapped volume into the multilamellar vesicles is determined from the difference between the maximal facilitated release and the total release after lysis of the liposomes by Triton X-100. The volume entrapped in the multilamellar vesicles determined this way amounted to 10–20% of the total entrapped volume.     

Liposome uptake into human colon adenocarcinoma cells in monolayer, spinner, and trypsinized cultures

The purpose of this study was to begin investigating the nature of liposome interactions with colon tumor cells. Thus, experiments were performed to study the uptake and incorporation of multilamellar and of reverse-phase evaporation liposomes of neutral charge into monolayers, suspended spinner cultures, and trypsinized cells of a human colon adenocarcinoma cell line, LS174T. The results showed that the same tumor cells cultured under each condition exhibited a distinct pattern of vesicle uptake as determined at 0, 15, 30, 60, and 120 min. In monolayer cultures of LS174T cells, the uptake of liposomes bearing [3H]actinomycin D in the lipid bilayers was linear throughout the incubation period. In contrast, in trypsinized and spinner suspension cultures, uptake of liposomes was biphasic. There was a proportional uptake of both liposome (labeled with [3H]phosphatidylcholine or [14C]cholesterol) and of actinomycin D (trace labeled with 3H) into the cells under all culture conditions, indicating quantitative delivery of the drug with the intact lipid vesicle. Although the amount of actinomycin D presented to tumor cells by the two liposomes was equivalent, reverse-phase evaporation liposomes were more effective than multilamellar vesicles in inhibiting uridine uptake. In the presence of excess liposomes (10 times the uptake studies), saturation of the tumor cell surface occurred by 120 min. However, the liposomes remained accessible to enzymatic removal for 60 min. Liposome-saturated tumor cells remained refractory to further binding of liposomes for at least 2 hr. The results thus revealed that differences in cell uptake were due to the state of the target cells and not the liposome types, or their differential leakage of labels.     

Surface areas of lipid membranes

Upon photolysis, alkyl pentacyanocobaltate complexes generate alkyl radicals which react rapidly and specifically with nitroxide radicals, and which do not penetrate phospholipid bilayers. By measuring the loss of paramagnetic resonance signal intensity when multilamellar liposomes containing a small amount of spin-labeled lipid are exposed to these radicals, we have measured the proportion of lipid on the external surface of liposomes. We have shown that liposomes prepared under specified conditions from dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and binary mixtures of dipalmitoylphosphatidylcholine and cholesterol all have the same proportion of external lipid.