Unilamellar Dimyristoyl Phosphatidylcholine Liposomes Research Articles

The THE INFLUENCE OF CHLORPROMAZINE HYDROCHLORIDE ON THE SIZE OF SMALL UNILAMELLAR DIMYROSTYLPHOSPHATIDYLCHOLINE LIPOSOMES AS REVEALED BY LIGHT SCATTERING PHOTOMETRY

Objectives: This study aims to investigate the possible influence of the model, cationic, surface-active solute chlorpromazine hydrochloride (CPZ-HCl) on the size of small unilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes as a function of temperature and CPZ-HCl concentration, below and above the critical micelle concentration (CMC).
 Methods: Small unilamellar DMPC liposomes were prepared by dissolving DMPC in chloroform and the solvent was rota-evaporated in a water bath adjusted at 40 °C. The lipid film was then dispersed in 0.1 M KCl solution adjusted at pH 6.2 to form large multilamellar liposomes which are then sonicated and fractionated via Sepharose 2B-Cl gel. The elution profile was followed spectrophotometrically at λ 260 nm. Combined fractions from the trailing edge of the included peak which is due to small unilamellar liposomes, were used as a source throughout this study. The SOFICA light scattering photometer (Model 42000) was used to determine the weight average liposomes weight (Lw) of small unilamellar DMPC liposomes. The Lw was determined in the absence and presence of CPZ-HCl both above and below the CMC over the temperature range of 25 °C to 40 °C.
 Results: The Lw was observed to increase linearly in the absence and presence of CPZ-HCl.
 The Lw was observed to increase linearly in the absence of CPZ-HCl, from 1.88×106+0.02 g/mol at 25 °C to 3.25×106+0.03 g/mol at 40 °C. Similarly, the Lw was observed to increase linearly in the present of CPZ-HCl, for example at 18 mmol drug concentration, the Lw increases from 11×106+0.04 g/mol at 25 °C to 13.75×106+0.03 g/mol at 40 °C. When the data are presented as a function of CPZ-HCl concentration, a gradual increase in Lw was observed below the CMC. Little increase in Lw however, was observed at post-micellar concentrations of 14 mmol and 18 mmol.

The effect of intercalants on the host liposome

When phospholipids are vigorously dispersed in water, liposomes are formed. In the present study, we have explored the effect of intercalant concentration on various properties of unilamellar liposomes. Liposomes were sonically intercalated with vitamin E acetate (VitEAc) and hypericin (Hy) until no difference in light transmission was observed, which reflects the formation of liposomes of minimal diameter. Our studies indicate that the intercalant structure and concentration have an influence on the liposome diameter, which could be directly measured by cryogenic transmittance electronic microscopy. Thus, intercalated VitEAc substantially decreased the diameter of unilamellar dimyristoylphosphatidylcholine liposomes, whereas Hy did not. In addition, we followed peak intensities in the absorbance and fluorescence spectra of Hy as a function of intercalant concentration in the liposomal solution. Initially, the fluorescence intensity increased linearly with concentration; however, the curve then arched asymptotically, followed by a decrease in fluorescence at yet higher concentrations. Because the Hy monomer is the only species that emits fluorescence, we believe that the decrease of fluorescence intensity is the result of Hy aggregation.

Interaction Free Energies of Eight Sodium Salts and a Phosphatidylcholine Membrane

Many recent reports have discussed specific effects of anions on the properties of lipid membranes and possible roles of such effects within biochemistry. One key parameter in both theoretical and experimental treatments of membrane-salt interactions is the net affinity, that is, the free energy of interaction. This parameter has been analyzed theoretically and by modeling of experimental data but not directly measured even for simple salt-membrane systems. To provide such information, we have studied the interaction of unilamellar liposomes of dimyristoyl phosphatidylcholine (DMPC) and eight sodium salts by dialysis equilibrium measurements. This method provides model free thermodynamic data and allows investigations in the dilute concentration range where solution nonideality and perturbation of membrane structure is limited. The transfer free energy of DMPC from water to salt solutions, Δμ(2,trans), was found to scale proportionally to the salt concentration, m(3), in the 0-50 mmolal range, and we suggest that the slope, Δμ(2,trans)/(out)m(3), represents an experimental salt-membrane interaction potential. The values of Δμ(2,trans)/(out)m(3) for sodium salts of monovalent anions ranged from +2.2 kJ (mol·molal)(-1) for F(-) to -16.9 kJ (mol·molal)(-1) for SCN(-), thus confirming a distinct membrane specificity among the anions. For Na(2)SO(4), the interaction was strongly unfavorable with Δμ(2,trans)/(out)m(3) = +20.6 kJ (mol·molal)(-1). The dialysis data were supplemented with calorimetric measurements and analyzed with respect to a two-domain model.

The effects of the natural antioxidant quercetin and anions of the Hofmeister series on liposomes marked with chlorophyll a

The unilamellar liposomes of dimyristoylphosphatidylcholine (DMPC), marked with chlorophyll a (Chla), have been chosen as suitable models in studies aimed at determining the effects of natural antioxidant quercetin (QCT) and Hofmeister series anions on lipid bilayers. The variations of steady state fluorescence emission intensity of Chla have been recorded and the values of Chla fluorescence anisotropy, under constant temperature and viscosity, at pH = 7.3-7.4 have been measured. Two types of experiments have been performed. In the first type of experiment, the concentration of anions was maintained constant and the concentration of QCT was varied (from 0 to 100 micromol/l). In the second type of experiment, the concentration of QCT was constant (30 micromol/l) and the concentration of anions was varied (from 0 to 152 mmol/l). The quenching of Chla fluorescence by QCT pleads in favor of QCT insertion at interface water-lipid, in the vicinity of the polar heads of lipids from liposomal bilayer at physiological pH and temperature. Fluorescence anisotropy of Chla in liposomes brought more evidences for QCT localizations at lipid/water interface. Chla is sensing a more rigid microenvironment when QCT is added to the lipid bilayer and specific effects of the Hofmeister series anions.

Thermodynamics of alcohol–lipid bilayer interactions: application of a binding model

Several recent reports have provided evidence that interactions of small alcohols with lipid bilayer membranes are dominated by adsorption to the membrane–water interface. This mode of interaction is better modeled by binding models than solution theories. In the present study, alcohol–membrane interactions are examined by applying the ‘solvent exchange model’ [J.A. Schellmann, Biophys. Chem. 37 (1990) 121] to calorimetric measurements. Binding constants (in mole fraction units) for small alcohols to unilamellar liposomes of dimyristoyl phosphatidylcholine were found to be close to unity, and in contrast to partitioning coefficients they decrease through the sequence ethanol, 1-propanol, 1-butanol. Thus, the direct (intrinsic) affinity of the bilayer for these alcohols is lower the longer the acyl chain. A distinction between binding and partitioning is discussed, and it is demonstrated that a high concentration of solute in the bilayer (large partitioning coefficients) can be obtained even in cases of weak binding. Other results from the model suggest that the number of binding sites on the lipid bilayer interface is 1–3 times the number of lipid molecules and that the binding is endothermic with an enthalpy change of 10–15 kJ/mol. Close to the main phase transition of the lipid bilayer the results suggest the presence of two distinct classes of binding sites: ‘normal’ sites similar to those observed at higher temperatures, and a lower number of high-affinity sites with binding constants larger by one or two orders of magnitude. The occurrence of high-affinity sites is discussed with respect to fluctuating gel and fluid domains in bilayer membranes close to the main phase transition.

A Calorimetric Investigation of the Interaction of Short Chain Alcohols with Unilamellar DMPC Liposomes

The molecular mechanisms by which ethanol and other short-chain alcohols perturb biological systems have been suggested to involve specific interactions with proteins and/or nonspecific interactions with the lipid bilayer part of biological membranes. To gain insight into the influence of alcohols on well-defined lipid bilayers, isothermal titration calorimetry (ITC) is used to determine the excess partial molar enthalpy of alcohol in liposome suspensions as well as the excess partial molar enthalpy of liposomes in dilute alcohol−water mixtures at temperatures above the main phase transition of dimyristoyl phosphatidylcholine (DMPC). The alcohols investigated were ethanol, 1-propanol, and 1-butanol. The calorimetric data were used to evaluate molecular interactions and based on a simple partitioning model the standard molar enthalpy of transferring alcohol from water into unilamellar DMPC liposomes was determined. The results reveal that the interaction of short-chain alcohols with the DMPC lipid bilayer ...

The topography of acetylcholinesterase in dimyristoylphosphatidylcholine liposomes

Unilamellar liposomes prepared from sn-3-(dimyristoyl)phosphatidylcholine (DMPC) in the presence and absence of acetylcholinesterase were examined by ESR for lipid/protein interactions. Using 5-, 12-, and 16-doxyl stearic acid probes incorporated into the phospholipid bilayers, no measurable differences in the gel to liquid-crystalline phase transition temperature of DMPC (as determined by ESR spectroscopy) were observed when the enzyme was present. These results have established that no significant incorporation of acetylcholinesterase into the hydrophobic region of the phospholipid bilayer is detectable at the protein: lipid ratios used in these experiments. Confirmation of these results was also obtained by differential scanning calorimetry. Interaction of the enzyme with the outermost region of the bilayer was established by trypsin digestion which indicated that as much as 25% of liposome-associated enzyme was removable and was, therefore, exposed to the outer surface. The results of this study have established that acetylcholinesterase associated with unilamellar DMPC liposomes was primarily entrapped within the aqueous compartment of the vesicles and was not present in the phospholipid bilayer.