dioleoyl-phosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylethanolamine

This study investigates the impact of the chemical nature of lipids and additive on the formulation and properties of pH sensitive liposomes. The objective is to understand the respective role of the formulation parameters on the liposome properties in order to optimize the conditions for efficient encapsulation of doxorubicin (DOX). These liposomes should be stable at physiological pH, and disrupt in slightly acidic media such as the tumor microenvironment to release their DOX load. The major challenge for encapsulating DOX in pH sensitive liposomes lies in the fact that this drug is soluble at low pH (when the pH-sensitive liposomes are not stable), but the DOX aqueous solubility decreases in the pH conditions corresponding to the stability of the pH-sensitive liposomes. The study of pH-sensitivity of liposomes was conducted using carboxyfluorescein (CF) encapsulated in high concentration, i.e. quenched, and following the dye dequenching as sensor of the liposome integrity. We studied the impact of (i) the chemical nature of lipids (dioleoyl phosphatidyl ethanolamine (DOPE), palmitoyl-oleoyl phosphatidyl ethanolamine (POPE) and dimyristoyl phosphatidyl ethanolamine (DMPE)) and (ii) the lipid/stabilizing agent ratio (alpha-tocopheryl succinate), on the pH sensitivity of the liposomes. Optimized liposome formulations were then selected for the encapsulation of DOX by an active loading procedure, i.e. driven by a difference in pH inside and outside the liposomes. Numerous experimental conditions were explored, in function of the pH gradient and liposome composition, which allowed identifying critical parameters for the efficient DOX encapsulation in pH-sensitive liposomes.

Topics: alpha-Tocopherol; Chemistry, Pharmaceutical; Doxorubicin; Fluoresceins; Hydrogen-Ion Concentration; Lipids; Liposomes; Phosphatidylethanolamines; Solubility; Tumor Microenvironment

Because transmembrane proteins (TMPs) can be obtained with sufficient purity for X-ray diffraction studies more frequently than decades ago, their mechanisms of action may now be elucidated. One of the pending issues is the actual interplay between transmembrane proteins and membrane lipids. There is strong evidence of the involvement of specific lipids with some membrane proteins, such as the potassium crystallographically sited activation channel (KcsA) of Streptomyces lividans and the secondary transporter of lactose LacY of Escherichia coli, the activities of which are associated with the presence of anionic phospholipids such as the phosphatidylglycerol (PG) and phosphatidyethanolamine (PE), respectively. Other proteins such as the large conductance mechanosensitive channel (MscL) of E. coli seem to depend on the adaptation of specific phospholipids to the irregular surface of the integral membrane protein. In this work we investigated the lateral compressibility of two homoacid phosphatidylethanolamines (one with both acyl chains unsaturated (DOPE), the other with the acyl chains saturated (DPPE)) and the heteroacid phosphatidyletanolamine (POPE) and their mixtures with POPG. The liquid expanded (LE) to liquid condensed (LC) transition was observed in POPE at a temperature below its critical temperature (T

Topics: Bacterial Proteins; Compressive Strength; Escherichia coli; Escherichia coli Proteins; Ion Channels; Microscopy, Atomic Force; Monosaccharide Transport Proteins; Phosphatidylethanolamines; Potassium Channels; Streptomyces lividans; Symporters; Temperature; Thermodynamics; Unilamellar Liposomes

In the search to uncover ethanol's molecular mechanisms, the calcium and voltage activated, large conductance potassium channel (BK) has emerged as an important molecule. We examine how cholesterol content in bilayers of 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE)/sphingomyelin (SPM) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) affect the function and ethanol sensitivity of BK. In addition, we examine how manipulation of cholesterol in biological membranes modulates ethanol's actions on BK. We report that cholesterol levels regulate the change in BK channel open probability elicited by 50 mM ethanol. Low levels of cholesterol (<20%, molar ratio) supports ethanol activation, while high levels of cholesterol leads to ethanol inhibition of BK. To determine if cholesterol affects BK and its sensitivity to ethanol through a direct cholesterol-protein interaction or via an indirect action on the lipid bilayer, we used the synthetic enantiomer of cholesterol (ent-CHS). We found that 20% and 40% ent-CHS had little effect on the ethanol sensitivity of BK, when compared with the same concentration of nat-CHS. We accessed the effects of ent-CHS and nat-CHS on the molecular organization of DOPE/SPM monolayers at the air/water interface. The isotherm data showed that ent-CHS condensed DOPE/SPM monolayer equivalently to nat-CHS at a 20% concentration, but slightly less at a 40% concentration. Atomic force microscopy (AFM) images of DOPE/SPM membranes in the presence of ent-CHS or nat-CHS prepared with LB technique or vesicle deposition showed no significant difference in topographies, supporting the interpretation that the differences in actions of nat-CHS and ent-CHS on BK channel are not likely from a generalized action on bilayers. We conclude that membrane cholesterol influences ethanol's modulation of BK in a complex manner, including an interaction with the channel protein. Finally, our results suggest that an understanding of membrane protein function and modulation is impossible unless protein and surrounding lipid are considered as a functional unit.

Topics: Biophysical Phenomena; Cholesterol; Ethanol; HEK293 Cells; Humans; Ion Channel Gating; Large-Conductance Calcium-Activated Potassium Channels; Lipid Bilayers; Microscopy, Atomic Force; Phosphatidylethanolamines; Phosphatidylserines; Sphingomyelins; Stereoisomerism

The energy associated with a mismatch between the hydrocarbon portions of a lipid bilayer and the hydrophobic regions of a transmembrane protein requires that one or both components deform in an attempt to minimize the energy difference. Transmembrane potassium channel subunits are composed of different structural motifs, each responsible for ion-selectivity, conductance and gating capabilities. Each has an inherent degree of flexibility commensurate with its amino acid composition. It is not clear, however, how each structural motif will respond to a fixed amount of distortion applied to the whole structure. We examined the single-channel conductance (G(c)) and gating (open probability, P (o)) of single BK(Ca) channels (hslo alpha-subunits) inserted into planar lipid bilayers containing 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE) or DOPE with either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or sphingomyelin (SPM) and 1-palmitoyl-2-oleoyl-3-phosphatidylethanolamine (POPE) with SPM. These latter three binary mixtures formed stable membranes with different distributions of thickness domains as determined by atomic force microscopy. Channels placed in each composition should be exposed to different amounts of distortion. BK(Ca) channels forced into the DOPE/SPM bilayer containing lipid domains with two different thicknesses showed two distinct levels of G(c) and P(o). The alterations in G(c) and P(o) were reciprocal. A larger conductance was accompanied by a smaller value for gating and vice versa. Channels forced into the POPE/SPM bilayer containing lipid domains with different thicknesses showed more than two distinct levels of G(c) and P(o). Channels placed in a uniform bilayer (DOPE/DOPC) showed a uniform distribution of conductance and activation. We conclude that both the inner and outer domains of the channel where these two channel functions are localized respond to deformation and that a fixed amount of distortion results in reciprocal changes in protein function.

Topics: Cell Line; Humans; Hydrophobic and Hydrophilic Interactions; Ion Channel Gating; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Lipid Bilayers; Microscopy, Atomic Force; Phosphatidylcholines; Phosphatidylethanolamines; Sphingomyelins

Nisin, a peptide used as a food preservative, is shown, by 31P-nuclear magnetic resonance and infrared spectroscopy, to perturb the structure of membranes formed of unsaturated phosphatidylethanolamine (PE) and to induce the formation of inverted non-lamellar phases. In the case of dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), nisin promotes the formation of inverted hexagonal phase. Similarly, the peptide induces the formation of an isotropic phase, most likely a cubic phase, with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE). It is proposed that the insertion of the peptide in the bilayer shifts the amphiphilic balance by increasing the hydrophobic contribution and is at the origin of the changes in the polymorphic propensities of PE. This is supported by the fact that the presence of cholesterol in the PE bilayer inhibits the power of nisin to perturb the membrane structure, most likely because the peptide insertion is difficult in the fluid ordered phase. This finding provides insight into possible antibacterial mechanisms of nisin.

Topics: Amino Acid Sequence; Food Preservatives; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Nisin; Phosphatidylethanolamines; Spectrophotometry, Infrared

Conformational disorder in liquid alkenes and in the L alpha and Hparallel phases of some unsaturated phospholipids has been monitored by FTIR spectroscopy. The CH2 wagging region (1330-1390 cm-1) in saturated chains contains vibrations of particular 2- and 3-bond conformational states as follows: 1341 cm-1, end-gauche (eg); 1352 cm-1, double gauche (gg); 1368 cm-1, the sum of kink and gtg states. In unsaturated chains, this spectral region revealed an additional band at 1362 cm-1 and (occasionally) a feature near 1348 cm-1. The 1362 cm-1 band is tentatively assigned to the wagging of CH2 groups adjacent to the C = C bond. Substantial populations of both gg and (kink+gtg) states are evident in the L alpha phases of unsaturated phosphatidylcholines (PC's). Unsaturated phosphatidylethanolamines (PE's) are more ordered than their PC counterparts, and possess fewer gg and eg states. Chain disorder in the Hparallel phase of PE's approaches that in L alpha phases of unsaturated PC's. Changes in conformer distributions during the L alpha-->Hparallel transition in 1,2-dioleoylphosphatidylethanolamine (DOPE), 1-palmitoyl,2-oleoylphosphatidylethanolamine (POPE), 1,2-dielaidoylphosphatidylethanolamine (DEPE), and N-methyl-DOPE(N-MeDOPE) were semi-quantitatively estimated. For DOPE and DEPE, slight cooperative increases in both gg and (kink+gtg) states occur, for POPE only the gg population increases and for N-MeDOPE only the kink+gtg populations increase. These disorder increases are consistent with the small calorimetric delta H for this transition. Difficulties in quantitative determination of conformational disorder in unsaturated chains are discussed.

Topics: Alkenes; Gas Chromatography-Mass Spectrometry; Magnetic Resonance Spectroscopy; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Spectroscopy, Fourier Transform Infrared; Thermodynamics