Liposomes: preparation, characterization, and preservation.
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Drug Stability↗
Biomedical subjects
D Lichtenberg
Publications and source records attributed to D Lichtenberg.
On the solubility of calcium deoxycholate: kinetics of precipitation and the effect of conjugated bile salts and lecithin.
In view of the low solubility of calcium deoxycholate and the possible induction of cholesterol precipitation in the gallbladder by calcium insoluble salts, we find it of interest to study the precipitation of calcium deoxycholate and its dependence on other bile components. The findings of these studies were as follows: (i) Precipitation of calcium deoxycholate from mixtures of calcium chloride and monomeric deoxycholate (at concentrations below the critical micelle concentration (CMC] is very slow even at relatively high CaCl2 concentrations (more than 20 days at 50 mM CaCl2). (ii) At higher deoxycholic acid (DOC) concentrations, precipitation of micellar DOC is faster and requires much lower calcium chloride concentrations. For any given calcium concentration, the rate of precipitation is maximal at an optimal DOC concentration. In solutions containing 150 mM NaCl, the maximal rate of precipitation occurs at about 10 mM DOC, almost independent of Ca2+ concentration. At lower ionic strength (10 mM NaCl), the optimal DOC concentration is 30 mM. These observations suggest that the most important factors in determining the rate of Ca(DOC)2 precipitation are (a) the ratio between calcium ions bound to the surface of a DOC micelle, and the [DOC] (the Ca2+/DOC binding ratio) and (b) the concentration of DOC micelles. (iii) In the presence of conjugated deoxycholates, the crystallization of calcium deoxycholate is inhibited. Phosphatidylcholine has a similar, although smaller, inhibitory effect. Upon precipitation of calcium deoxycholate from a mixed micellar system containing sodium deoxycholate, phosphatidylcholine and cholesterol, the latter two components spontaneously form vesicles. The anti-nucleating effect of PC and conjugated bile salts is explained in terms of "poisoning" of the crystallization process. In view of the latter results we conclude that under normal conditions calcium deoxycholate is not likely to precipitate in the gallbladder.
Bile Acids and Salts↗
Precipitation of calcium palmitate from bile salt-containing dispersions.
Addition of calcium chloride to mixed micellar systems composed of sodium salts of palmitic acid and high concentrations of different bile acids results in precipitation of Ca(palmitate)2 only when the palmitate concentration exceeds a critical value, which is dependent on the concentrations of Ca2+, Na+ and bile salt, and on the type of bile salt used. All these dependencies, as well as the complex and interrelated effects of the various parameters on the kinetics of Ca(palmitate)2 precipitation are consistent with the following mechanism: (i) calcium binds to palmitate-bile salt mixed micelles and promotes their aggregation, at a rate governed by the concentration ratio between bound calcium and micelles (here denoted "binding ratio"). (ii) Ca(palmitate)2 precipitation occurs within the aggregate of micelles only if those micelles include sufficient amounts of Ca2+ and palmitate to allow for the formation of large enough crystal units of Ca(palmitate)2 which can serve as nucleation "seeds". Both the concentrations of micelles and Na+ have dual effects on the rate of precipitation. Increasing micelle concentration, by itself, accelerates aggregation but at the same time leads to a decrease of the binding ratio, thus reducing the rate of precipitation. Na+ which reduces the binding ratio through competitive binding also reduces the surface charge, thus assisting micelle aggregation. Our model also explains the facilitation of precipitation observed when phosphatidylcholine is contained in the palmitate-bile salt mixed micelles and the inhibitory effect of the water soluble bovine serum albumin.
Bile Acids and Salts↗
Stability of mixed micellar bile models supersaturated with cholesterol.
The maximal equilibrium solubility of cholesterol in mixtures of phosphatidylcholine (PC)1 and bile salts depends on the cholesterol/PC ratio (Rc) and on the effective ratio (Re) between nonmonomeric bile salts and the sum (CT) of PC and cholesterol concentrations (Carey and Small, 1978; Lichtenberg et al., 1984). By contrast, the concentration of bile salts required for solubilization of liposomes made of PC and cholesterol does not depend on Rc (Lichtenberg et al., 1984 and 1988). Thus, for Rc greater than 0.4, solubilization of the PC-cholesterol liposomes yields PC-cholesterol-bile salts mixed micellar systems which are supersaturated with cholesterol. In these metastable systems, the mixed micelles spontaneously undergo partial revesiculation followed by crystallization of cholesterol. The rate of the latter processes depends upon Rc, Re, and CT. For any given Rc and Re, the rate of revesiculation increases dramatically with increasing the lipid concentration CT, reflecting the involvement of many mixed micelles in the formation of each vesicle. The rate also increases, for any given CT and Re, upon increasing the cholesterol to PC ratio, Rc, probably due to the increasing degree of supersaturation. Increasing the cholate to lipid effective ratio, Re, by elevation of cholate concentration at constant Rc and CT has a complex effect on the rate of the revesiculation process. As expected, cholate concentration higher than that required for complete solubilization at equilibrium yields stable mixed micellar systems which do not undergo revesiculation, but for lower cholate concentrations decreasing the degree of supersaturation (by increasing [cholate]) results in faster revesiculation. We interpret these results in terms of the structure of the mixed micelles; micelles with two or more PC molecules per one molecule of cholesterol are relatively stable but increasing the bile salt concentration may cause dissociation of such 1:2 cholesterol:PC complexes, hence reducing the stability of the mixed micellar dispersions. The instability of PC-cholesterol-cholate mixed systems with intermediary range of cholate to lipids ratio may be significant to gallbladder stone formation as: (a) biliary bile contains PC-cholesterol vesicles which may be, at least partially, solubilized by bile salts during the process of bile concentration in the gallbladder, resulting in mixtures similar to our model systems; and (b) the bile composition of cholesterol gallstone patients is within an intermediary range of bile salts to lipids ratio.
Cholesterol↗
Fusion of Sendai virus with negatively charged liposomes as studied by pyrene-labelled phospholipid liposomes.
Sendai virus particles fuse with negatively charged liposomes but not with vesicles made of zwitterionic phospholipids. The liposome-virus fusion process was studied by dilution of the concentration-dependent excimer-forming fluorophore 2-pyrenyldodecanoylphosphatidylcholine contained in the liposomes by the viral lipids. The data were analyzed in the framework of a mass action kinetic model. This provided analytical solutions for the final levels of probe dilution and numerical solutions for the kinetics of the overall fusion process, in terms of rate constants for the liposome-virus adhesion, deadhesion and fusion. This analysis led to the following conclusions: At neutral pH and 37 degrees C, only 15% of the virus particles can fuse with the phospholipid vesicles, although all the virions may aggregate with the liposomes. The rate constants for aggregation, fusion and deadhesion are of the orders of magnitude of 10(7) M-1 X s-1, 10(-3) s-1 and 10(-2), s-1, respectively. The fraction of active virus increases with temperature. At acidic pH, both the fraction of 'fusable' virus and the rate of fusion increase markedly. The optimal pH for fusion is 3-4, where most of the virus particles are active. At higher pH values, an increasing fraction of the virus particles become inactive, probably due to ionization of viral glycoproteins, whereas at pH values below 3.0 the fusion is markedly reduced, most likely due to protonation of the negatively charged vesicles. While only 15% of the virions fuse with the liposomes at pH 7.4 and 37 degrees C, all the liposomes lose their content (Amselem, S., Loyter, A. Lichtenberg, D. and Barenholz, Y. (1985) Biochim. Biophys. Acta 820, 1-10). We therefore propose that release of entrapped solutes is due to liposome-virus aggregation, and not to fusion. Both trypsinization and heat inactivation of the virus particles inhibit not only the fusion process but also the release of carboxyfluorescein. This demonstrates the obligatory role of viral membrane proteins in liposome-virus aggregation. Reconstituted vesicles made of the viral lipid and the hemagglutinin/neuraminidase (HN) glycoprotein fuse with negatively charged liposomes similar to the intact virions. This suggests that the fusion of virions with negatively charged vesicles, unlike the fusion of the virus with biological membranes, requires only the HN and not the fusion glycoprotein.
Calcium↗
Kinetic and structural aspects of reconstitution of phosphatidylcholine vesicles by dilution of phosphatidylcholine-sodium cholate mixed micelles.
Dilution of mixed micellar dispersions of egg phosphatidylcholine (PC) and sodium cholate beyond a critical value results in formation of cholate-containing PC vesicles. The structure of the resultant vesicles and some mechanistic aspects of this process have been investigated by the use of light scattering and nuclear magnetic resonance techniques. The main findings and conclusions are the following: Both the state of aggregation (micellar or vesicular) and the apparent equilibrium size distribution of micelles or vesicles obtained by dilution of the PC-cholate mixed micellar dispersions are a function of the cholate to PC molar ratio in the mixed aggregates (micelles or vesicles). When this effective ratio (Re) is higher than 0.4, the dispersion is micellar, and the size of the mixed micelles increases with decreasing Re; when Re less than 0.3, the dispersion is essentially vesicular, and the mean hydrodynamic radius of the vesicles is an increasing function of Re; in dispersions with 0.3 less than Re less than 0.4, mixed micelles and vesicles coexist. Addition of cholate to vesicular dispersions, to Re values below 0.3, results in vesicle size growth through a concentration-independent lipid-exchange mechanism. Addition of cholate to higher Re values results in micellization (solubilization) of the vesicles. On the other hand, dilution of vesicular dispersions does not affect the size of the vesicles. Apparent equilibration of a mixed micellar dispersion following dilution to Re values below 0.3 is slow (many hours). The overall process involves a series of three subsequent categories of steps: (i) a rapid (approximately 1-2 min) prevesiculation equilibration of micellar sizes.(ABSTRACT TRUNCATED AT 250 WORDS)
Cholic Acid↗
Hydrolysis of dipalmitoylphosphatidylcholine small unilamellar vesicles by porcine pancreatic phospholipase A2.
The hydrolysis of small unilamellar vesicles made of dipalmitoylphosphatidylcoline by pancreatic phospholipase A2 has been studied under various conditions of temperature and enzyme and substrate concentration using the following three different experimental protocols. When the enzyme was added to the substrate vesicles after being separately adjusted to the temperature of the experiments hydrolysis occurred instantaneously only in the temperature range where the lipid is known to exist in its gel phase, while above the transition range no hydrolysis occurred. Within the transition range, the time course of hydrolysis was characterized by initial very slow rate of hydrolysis (latency phase) followed by an abrupt increase in the rate after a time tau, which is a complex function of temperature and enzyme to substrate ratio. When an enzyme-substrate mixture was first preincubated below Tm and then temperature jumped to a temperature above or within the transition range, the latency phase was markedly shortened. When the temperature jump was to the transition range, this effect is observed even if Ca2+ is absent in the preincubation mixture. However, instantaneous hydrolysis was observed upon temperature jumping the mixture to a temperature high above Tm only if Ca2+ was present in the preincubation medium. In temperature-scanning experiments, hydrolysis was followed while changing the temperature of the enzyme-substrate mixture continuously. Heating an enzyme-substrate mixture from room temperature resulted in an abrupt onset of hydrolysis when the transition range was approached. These results lead us to conclude that two distinctly different steps precede rapid hydrolysis of dipalmitoylphosphatidylcholine small unilamellar vesicles by pancreatic phospholipase A2: a Ca2+-independent binding of the enzyme to the substrate vesicles, which for chemically pure bilayers occurs best in the gel phase. This step is followed by a Ca2+-dependent activation of the initially formed enzyme-substrate complex. The latter step only occurs under conditions where the bilayer possesses packing irregularities and probably involves a reorganization of the enzyme-substrate complex. At least one of these two steps appears to involve enzyme-enzyme interaction.
Animals↗
Hydrolysis of dipalmitoylphosphatidylcholine large unilamellar vesicles by porcine pancreatic phospholipase A2.
The interaction between dipalmitoylphosphatidylcholine large unilamellar vesicles and porcine pancreatic phospholipase A2 has been studied under a variety of conditions. It was found that the presence of large unilamellar vesicles inhibits the hydrolysis of small unilamellar vesicles at room temperature, and reaction calorimetric experiments showed that protein-lipid interactions in the absence of Ca2+ occur in the gel state with a stoichiometry of about 40 phospho-lipid molecules/protein-binding site. However, hydrolysis can be induced in the gel state under conditions of osmotic shock. On the other hand, hydrolysis is usually observed within the lipid transition temperature range, but then it occurs only after a latency phase during which the hydrolysis is very slow. The duration of this latency phase reaches a minimum near the phase transition temperature. However, if the enzyme-substrate mixture is heated from low temperatures (continuously or by a temperature jump) to a temperature within the phase transition region, hydrolysis occurs instantaneously. These results are in accordance with the conclusions of the preceding paper (Menashe, M., Romero, G., Biltonen, R. L., and Lichtenberg, D. (1986) J. Biol. Chem. 261, 5328-5333) that effective binding of the enzyme to lipid vesicles occurs relatively rapidly in the gel state and that activation of the enzyme-substrate complex requires the existence of structural irregularities in the lipid bilayer. Although hydrolysis products may have a pronounced effect on the time course of the reaction in the transition range, instantaneous hydrolysis can be induced in the phase transition region in the absence of reaction products by appropriate manipulation of the experimental conditions during which no reaction products are produced. Thus reaction products are not essential for activation of porcine pancreatic phospholipase A2. Furthermore, it is shown that the fraction of lipid hydrolyzed during the latency period is a function of the initial substrate concentration in a manner inconsistent with the proposition that the accumulation of a constant critical fraction of reaction products is the basis for activation. Comparison of the results of this study with those of the preceding paper strongly support the previously proposed reaction scheme.
Animals↗
Characterization of the solubilization of lipid bilayers by surfactants.
This communication addresses the state of aggregation of lipid-detergent mixed dispersions. Analysis of recently published data suggest that for any given detergent-lipid mixture the most important factor in determining the type of aggregates (mixed vesicles or mixed micelles) and the size of the aggregate is the detergent to lipid molar ratio in these aggregates, herein denoted the effective ratio, Re. For mixed bilayers this effective ratio has been previously shown to be a function of the lipid and detergent concentrations and of an equilibrium partition coefficient, K, which describes the distribution of the detergent between the bilayers and the aqueous phase. We show that, similar to mixed bilayers, the size of mixed micelles is also a function of the effective ratio, but for these dispersions the distribution of detergent between the mixed micelles and the aqueous medium obeys a much higher partition coefficient. In practical terms, the detergent concentration in the mixed micelles is equal to the difference between the total detergent concentration and the critical micelle concentration (cmc). Thus, the effective ratio is equal to this difference divided by the lipid concentration. Transformation of mixed bilayers to mixed micelles, commonly denoted solubilization, occurs when the surfactant to lipid effective ratio reaches a critical value. Experimental evaluation of this critical ratio can be based on the linear dependence of detergent concentration, required for solubilization, on the lipid concentration. According to the 'equilibrium partition model', the dependence of the 'solubilizing detergent concentration' on the lipid concentration intersects with the lipid axis at -1/K, while the slope of this dependence is the critical effective ratio. On the other hand, assuming that when solubilization occurs the detergent concentration in the aqueous phase is approximately equal to the critical micelle concentration, implies that the above dependence intersects with the detergent axis at the critical micelle concentration, while its slope, again, is equal to the critical effective ratio. Analysis of existing data suggests that within experimental error both these distinctively different approaches are valid, indicating that the critical effective ratio at which solubilization occurs is approximately equal to the product of the critical micelle concentration and the distribution coefficient K. Since the nature of detergent affects K and the critical micelle concentration in opposite directions, the critical ('solubilizing') effective ratio depends upon the nature of detergent less than any of these two factors.
Kinetics↗
The interaction of Sendai virus with negatively charged liposomes: virus-induced lysis of carboxyfluorescein-loaded small unilamellar vesicles.
The interaction of Sendai virus with small, unilamellar vesicles, lacking virus receptors and loaded with self-quenched 6-carboxyfluorescein, was studied. Sendai virions induced release of carboxyfluorescein from vesicles composed of negative charged phospholipids, despite the fact that they did not contain virus receptors. Preliminary experiments indicate that the carboxyfluorescein release is accompanied by mixing of the virus and liposome lipids and their entrapped contents, suggesting liposome-virus fusion. No release of carboxyfluorescein was observed with vesicles containing only phosphatidylcholine. The rate of virus-induced carboxyfluorescein release was temperature dependent; the lytic activity of the virus was greatly enhanced above 25 degrees C. This effect was not due to a thermal phase transition of the lipids in either the lipid vesicles or the virions. Virus-induced carboxyfluorescein release was inhibited by the presence of calcium ions in the medium and of cholesterol in the lipid vesicles. It increased with increasing concentrations of either the lipid vesicles or the virions. pretreatment of virions with increasing concentrations of three different proteolytic enzymes (trypsin, chymotrypsin and proteinase) inhibited the virus' ability to cause release of carboxyfluorescein from negatively charged liposomes. Inhibition of the viral lytic activity was also observed after virions were incubated above 56 degrees C.
Calcium↗
Structural characteristics of phospholipid multilamellar liposomes.
The relative proportion of lipid on the external surface of spherical multilamellar vesicles and the aqueous volume trapped within them, can be computed as a function of a liposome's outer radius, interlamellar aqueous spacing, and the number of bilayers. When known experimental data is put into these calculations, the results lead to the conclusion that spontaneously formed liposomes are on the average composed of up to 10 lamellae, whose total thickness is approximately 0.1 micron, and traps an aqueous sphere whose average radius is approximately 0.5 micron. Most of the aqueous medium entrapped within the multi-bilayers is contained in the internal core of the liposome. When assuming spherical liposomes and using percent exposure data, this calculation overestimates the experimentally detected trapped volumes.
Chemical Phenomena↗
Thermodynamic characterization of the pretransition of unilamellar dipalmitoyl-phosphatidylcholine vesicles.
High-sensitivity differential scanning calorimetry of small unilamellar vesicles (SUV) made by sonication of dipalmitoylphosphatidylcholine (DPPC) reveals that the gel-liquid crystalline transition at 37 C is preceded by a pretransition at 28 C that is relatively slow (t1/2 approximately equal to 2-4 min) and has an enthalpy change of ca. 0.2 kcal/mol. On incubation at 4 C, these SUV fuse spontaneously into large unilamellar vesicles (LUV). LUV also exhibit both a pretransition and a main-phase transition. The temperature of the main transition (Tm) and the enthalpy change of both the pretransition and main transition of these fused vesicles are similar to those of large multilamellar vesicles (MLV). The enthalpy change associated with the transition at 28 C decreases in SUV in a manner directly correlated to the decrease in the apparent enthalpy change of the 37 C main transition, indicating that the smaller (low temperature) transition is indeed a pretransition that is an inherent property of SUV. Therefore, unilamellar vesicles of DPPC appear to exhibit a pretransition at a temperature that varies from 28 C for the small vesicles to 35 C for the much larger vesicles.
Calorimetry, Differential Scanning↗
Solubilization of phospholipids by detergents. Structural and kinetic aspects.
Most amphiphiles in biological membranes including phospholipids, steroids, and membrane proteins are insoluble amphiphiles and would form liquid crystals or insoluble precipitates alone in aqueous media. Detergents are soluble amphiphiles and above a critical concentration and temperature form micelles of various sizes and shapes. Much of the recent progress in studying the insoluble amphiphiles is due to the formation of thermodynamically stable isotropic solutions of these compounds in the presence of detergents. This process, which is commonly denoted as "solubilization,' involves transformation of lamellar structures into mixed micelles. The information available to date on the solubilization of phospholipids, which constitute the lipid skeleton of biomembranes, by the common detergents is discussed in this review, both with respect to the kinetics of this process and the structure of the various phospholipid-detergent mixed micelles formed. It is hoped that this discussion will lead to somewhat more useful, although still necessarily fairly empirical, approaches to the solubilization of phospholipids by detergents.
Animals↗
Kinetics of transfer of gangliosides from their micelles to dipalmitoylphosphatidylcholine vesicles.
Two aspects of the kinetics of transfer of ganglioside from micelles to dipalmitoylphosphatidylcholine vesicles have been examined: (i) The first aspect is the rate of transfer of ganglioside from micelles at very low ganglioside/phospholipid ratios. Under these conditions the rate of incorporation into vesicles is independent of the vesicle concentration, indicating that transfer occurs by diffusion of ganglioside molecules through the aqueous phase and not by collision of micelles and vesicles. The initial transfer of monosialoganglioside is slower (t 1/2 = 2 h) than that of trisialoganglioside (t 1/2 = 0.5 h). The rate of transfer decreases during the transfer process. This decrease in rate depends on the character of the micelles and not on the ganglioside content of acceptor vesicles. The initial rate of transfer decreases sharply with decreasing temperature. (ii) The second aspect is the rate of transfer of ganglioside from micelles to phospholipid vesicles at high ganglioside/phospholipid ratios. In the presence of excess ganglioside, the level of incorporation into vesicles saturates when the ganglioside content of the vesicles reaches 12-15 mol %. This saturation level is not markedly dependent on the number of sialic acid residues in the ganglioside.
Colloids↗
Correlation between the psychotropic potency of cannabinoids and their effect on the 1H-NMR spectra of model membranes.
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Cannabinoids↗
Solubilization of phosphatidylcholine bilayers by octyl glucoside.
The solubilization of large, unilamellar egg phosphatidylcholine vesicles by the nonionic detergent octyl glucoside (OG) was investigated by nuclear magnetic resonance (NMR), fluorescence anisotropy, turbidity, electron microscopy, and centrifugation followed by compositional analysis. The solubilization process is well described by the three-stage model previously proposed for other detergents. In stage I, the OG partitions between the bilayer and aqueous phases with a molar partition coefficient of 59 +/- 6. The presence of OG in the bilayers produces a small "fluidizing" effect, as indicated by changes in the NMR and fluorescence anisotropy parameters. A rearrangement that forms large mixed bilayers occurs in the latter part of stage I. Stage II, the conversion of detergent-saturated bilayers into mixed micelles, begins at a ratio of total OG concentration minus the critical micelle concentration to total phosphatidylcholine concentration of approximately 1.5 and continues until this ratio reaches about 3.0. The correction for the critical micelle concentration of the OG is necessary for comparison of experimental results obtained at different lipid concentrations. The mixed bilayer-mixed micelle interconversion is quantified by the centrifugation experiments and by 31P NMR. The agreement between the two methods is excellent. Advantages of the NMR method are discussed. In stage III, which was not studied in detail here, all of the phosphatidylcholine is present as mixed micelles. Evidence is presented that the various structures present in the dispersions are in equilibrium with one another during these experiments.
Fluorescence Polarization↗
Transition of a liquid crystalline phosphatidylcholine bilayer to the gel phase in a vesicle reduces the internal aqueous volume.
The liquid crystalline to gel phase transition in phospholipid bilayers is associated with a marked reduction in the area per phospholipid molecule. Geometric considerations based on published data suggest that this decrease in molecular area is accompanied by a reduction in the internal aqueous volume trapped within a unilamellar bilayer vesicle. This volume reduction, which depends upon the shape of the vesicle, is shown to be between 23 and 60 percent. We have observed a 25 to 30 percent reduction in the internal aqueous volume of unilamellar vesicles about 700 A in diameter formed from dipalmitoylphosphatidylcholine using the self-quenching of 6-carboxyfluorescein trapped within this compartment.
Fluoresceins↗
Vesicle- vesicle interactions in sonicated dispersions of dipalmitoylphosphatidylcholine.
The time course of the size transformation of sonicated small unilamellar dipalmitoylphosphatidylcholine vesicles at 23 degrees C has been followed with (31)P and (1)H nuclear magnetic resonance (NMR) spectroscopy. Comparison of these results with turbidity measurements indicates that vesicle aggregation, monitored by turbidity, and size transformation, followed by NMR, occur on distinctly different time scales. For lipid concentrations in the 5-50 mM range, aggregation takes place on a time scale of minutes, whereas size transformation takes many hours. Aggregation, unlike size transformation, can be reversed by increasing the temperature above the phospholipid phase transition temperature. Analysis of the (31)P NMR line shapes provides evidence for a model in which the small vesicles transform into the product vesicles [characterized by Schullery, S.E., Schmidt, C.F., Felgner, P., Tillack, T.W., & Thompson, T.E. (1980) Biochemistry 19, 3919] without producing significant amounts of intermediate-size vesicles. Kinetic analysis indicates that the size transformation is apparently second order. (1)H NMR data indicate that the rate of transformation is decreased if trivalent ions are added to the dispersions and also if the temperature is periodically increased above the transition temperature. Analysis of the latter experiment provides some evidence that vesicle aggregation is a necessary precursor to size transformation. It was also found that increasing the average vesicle size decreases the extent of transformation.
Kinetics↗