Phospholipid Vesicle Membranes Research Articles

Modulation of Density and Orientation of Amphiphilic DNA on Phospholipid Membranes. II. Vesicles

In the present series of papers, we describe the results of a systematic study on the anchoring of cholesterol-tagged oligonucleotides to phospholipid bilayers followed by membrane-assisted hybridization of the complementary strand in solution. This paper compares the behavior of two cholesteryl modified oligonucleotides, differing in the architecture and hydrophobicity of the lipophilic moiety, in the self-aggregation, hybridization, and insertion in phospholipid vesicle membranes. We have focused our attention on a singly substituted derivative (SC-ON(1)) and a multicholesterol (MC-ON(1)) derivative, where the cholesteryl units are inserted at the desired positions along a noncoupling T-sequence. The self-aggregation properties in solution are also explicitly taken into account and evaluated as competitive with respect to the adsorption at fluid or solid interfaces and to hybridization with the complementary ON(2) sequence. By exploring a wide range of ON derivative concentrations, different peculiar scenarios emerge for different hydrophobicity of the amphiphilic DNA guest molecules on the vesicles, in terms of distribution and conformation of the single strand and consequent coupling properties with the complementary strand in solution.

Confocal Raman microscopy for simultaneous monitoring of partitioning and disordering of tricyclic antidepressants in phospholipid vesicle membranes

AbstractCharacterization of drug–membrane interactions is important in order to understand the mechanisms of action of drugs and to design more effective drugs and delivery vehicles. Raman spectra provide compositional and conformational information of drugs and lipid membranes, respectively, allowing membrane disordering effects and drug partitioning to be assessed. Traditional Raman spectroscopy and other widely used bioanalytical techniques such as differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) typically require high sample concentrations. Here, we describe how temperature‐controlled, optical‐trapping confocal Raman microscopy facilitates the analysis of drug–membrane interactions using micromolar concentrations of drug, while avoiding drug depletion from solution by working at even lower lipid concentrations. The potential for confocal Raman microscopy as an effective bioanalytical tool is illustrated using tricyclic antidepressants (TCAs), which are cationic amphiphilic molecules that bind to phospholipid membranes and influence lipid phase transitions. The interaction of these drugs with vesicle membranes of differing head‐group charge is investigated while varying the ring and side‐chain structure of the drug. Changes in membrane structure are observed in Raman bands that report intra‐ and intermolecular order versus temperature. The partitioning of drugs into the membrane can also be determined from the Raman scattering intensities. These results demonstrate the usefulness of confocal Raman microscopy for the analysis of drug–membrane systems at biologically relevant drug concentrations. Effective tools for monitoring drug–membrane interactions are crucial for rational design of new drugs. Copyright © 2009 John Wiley & Sons, Ltd.

Effect of Functional Groups on Incorporation Behavior of Amino Acid-Type Surfactant into Phospholipid Vesicle Membrane

The incorporation behaviors of some N-dodecanoyl amino acid-type surfactants into L-alpha-dipalmitoylphosphatidylcholine (DPPC) vesicles in aqueous solution were investigated. From the leakage measurements of a vesicle-entrapped fluorescence probe, it was found that these surfactants did not affect the DPPC vesicle so much at a very low concentration less than a one-tenth of the critical micelle concentration (CMC), but caused a significant release of the probe form the vesicles even at just below the CMC. The leakage induced by amino acid-type surfactants was promoted by increasing hydrophobicity of the amino acid. However the polarization of DPH embedded in the DPPC membrane was almost unchanged by incorporation of the surfactants at the concentration below the CMC except for the N-dodecanoylphenylalanine system. The binding enthalpy and the binding constant of the surfactant to DPPC vesicles were estimated by isothermal titration calorimetry. While the binding enthalpy was independent of the kind of surfactant, the binding constant increases with increasing hydrophobicity of the amino acid species in the surfactants. The molar ratio of bound surfactant to DPPC molecule in the saturated state was much greater than that for sodium dodecyl sulfate system. Further the ratio was not influenced by the structure of the amino acid side chain.

A Light-Gated Synthetic Ion Channel

A gated synthetic ion channel with beta-cyclodextrin as the pore and azobenzene as the gate is reported. Irradiation converts a tethered trans-azobenzene to cis-azobenzene which likely transforms the channel from a self-inclusion complex to a dissociated structure. This transformation results in an increase in anion transport and a decrease in cation transport across a phospholipid vesicle membrane.

Agglutination of like-charged red blood cells induced by binding of β2-glycoprotein I to outer cell surface

Plasma protein-mediated attractive interaction between membranes of red blood cells (RBCs) and phospholipid vesicles was studied. It is shown that β 2 -glycoprotein I ( β 2 -GPI) may induce RBC discocyte–echinocyte–spherocyte shape transformation and subsequent agglutination of RBCs. Based on the observed β 2 -GPI-induced RBC cell shape transformation it is proposed that the hydrophobic portion of β 2 -GPI molecule protrudes into the outer lipid layer of the RBC membrane and increases the area of this layer. It is also suggested that the observed agglutination of RBCs is at least partially driven by an attractive force which is of electrostatic origin and depends on the specific molecular shape and internal charge distribution of membrane-bound β 2 -GPI molecules. The suggested β 2 -GPI-induced attractive electrostatic interaction between like-charged RBC membrane surfaces is qualitatively explained by using a simple mathematical model within the functional density theory of the electric double layer, where the electrostatic attraction between the positively charged part of the first domains of bound β 2 -GPI molecules and negatively charged glycocalyx of the adjacent RBC membrane is taken into account.

Permeation of a β-heptapeptide derivative across phospholipid bilayers

Based on a number of experiments it is concluded that the fluorescein labeled β-heptapeptide fluoresceinyl-NH-CS- (S)- β 3hAla- (S)- β 3hArg- (R)- β 3hLeu- (S)- β 3hPhe- (S)- β 3hAla- (S)- β 3hAla- (S)- β 3hLys-OH translocates across lipid vesicle bilayers formed from DOPC (1,2-dioleoyl- sn-glycero-3-phosphocholine). The conclusion is based on the following observations: (i) addition of the peptide to the vicinity of micrometer-sized giant vesicles leads to an accumulation of the peptide inside the vesicles; (ii) if the peptide is injected inside individual giant vesicles, it is released from the vesicles in a time dependent manner; (iii) if the peptide is encapsulated within sub-micrometer-sized large unilamellar vesicles, it is released from the vesicles as a function of time; (iv) if the peptide is submitted to immobilized liposome chromatography, the peptide is retained by the immobilized DOPC vesicles. Furthermore, the addition of the peptide to calcein-containing DOPC vesicles does not lead to significant calcein leakage and vesicle fusion is not observed. The finding that derivatives of the β-heptapeptide (S)- β 3hAla- (S)- β 3hArg- (R)- β 3hLeu- (S)- β 3hPhe- (S)- β 3hAla- (S)- β 3hAla- (S)- β 3hLys-OH can translocate across phospholipid bilayers is supported by independent measurements using Tb 3+-containing large unilamellar vesicles prepared from egg phosphatidylcholine and wheat germ phosphatidylinositol (molar ratio of 9:1) and a corresponding peptide that is labeled with dipicolinic acid instead of fluorescein. The experiments show that this dipicolinic acid labeled β-heptapeptide derivative also permeates across phospholipid bilayers. The possible mechanism of the translocation of the particular β-heptapeptide derivatives across the membrane of phospholipid vesicles is discussed within the frame of the current understanding of the permeation of certain oligopeptides across simple phospholipid bilayers.

Transport of chloride and carboxyfluorescein through phospholipid vesicle membranes by heptapeptide amphiphiles.

Seven synthetic anion transporters (SAT) of the general form R(2)N-COCH(2)OCH(2)CO-(Gly)(3)-Pro-(Gly)(3)-OR' were prepared. Three pairs of compounds each contained twin n-hexyl, n-decyl, and n-octadecyl (R) groups at the N-terminus and one contained twin n-tetradecyl groups. Three of the compounds were C-terminated by benzyl and three by heptyl (R') residues. The ability of these compounds to mediate ion release from phospholipid vesicles was assessed. Chloride release was measured by ion selective electrode measurements and by chloride quenching of the fluorescent dye lucigenin. Transport of the anion carboxyfluorescein (CF) was measured by fluorescence dequenching. Differences in both the C- (R') and N-terminal (R) residues within the ionophores affected anion transport. The chloride release data acquired by ion selective electrode and fluorescence methods were similar but not identical. A possible carrier mechanism for Cl(-) transport was discredited. Both Cl(-) and CF anions were released from vesicles by these compounds. The results of CF and Cl(-) transport showed good consistency when the ionophore's N-terminal chains were either decyl or octadecyl but not when they were hexyl. The transport of CF and Cl(-) appears to be fundamentally different when R is C(6) compared to C(10) or C(18). Differences between the behavior of SATs with Cl(-) and CF were also reflected in negative ion mass spectrometric studies.

Detection of Drug−Membrane Interactions in Individual Phospholipid Vesicles by Confocal Raman Microscopy

Optical-trapping confocal Raman microscopy is developed as a method to study the interactions of drugs or other compounds with the membranes of individual phospholipid vesicles. This technique allows membrane disorder, permeability, and drug localization to be assessed without the need for labeling of the membrane or the compounds of interest. We have applied this technique to study the interactions of two nonsteroidal antiinflammatory drugs, salicylate and ibuprofen, with vesicles prepared from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The results show that both salicylate and ibuprofen increase membrane disorder, as determined from increases in the Raman scattering from gauche conformers in the phospholipid acyl chains. By monitoring the Raman scattering of the drug molecules in optically trapped DMPC vesicles, the membrane permeability and partitioning of the drugs could be determined; the spatial distributions of the drugs were also measured by scanning the laser focus through surface-adhered 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles, producing a profile of the vesicle and its contents. Though the membrane is permeable to both drugs, ibuprofen preferentially accumulates in the membrane, whereas salicylate does not. The measured ibuprofen accumulation agrees quantitatively with the water/octanol partition coefficient of the drug and the estimated volume of the lipid membrane. The results suggest that ibuprofen localizes in the hydrophobic acyl chain region of the membrane, whereas salicylate weakly associates with the phospholipid headgroups.

Longitudinal diffusion behavior of hemicyanine dyes across phospholipid vesicle membranes as studied by second-harmonic generation and fluorescence spectroscopies

The adsorption and longitudinal diffusion behaviors of a series of hemicyanine dyes to phospholipid vesicle membranes were studied by second-harmonic generation (SHG) and fluorescence spectroscopies. It was observed that the longitudinal diffusion of cationic hemicyanine dyes takes place immediately after the initial adsorption of these dyes to the outer surface of the vesicle membrane. In contrast, hardly any amount of a zwitterionic hemicyanine dye with a sulfonate group diffused across the vesicle membrane within the measurement time (<2000 s). Based on the difference in the time-course responses of SHG and fluorescence spectroscopies for all of the hemicyanine dyes tested, we propose that hydration of the sulfonate group is mainly responsible for the low diffusivity of the zwitterionic hemicyanine dye.

Unique incorporation behavior of amino acid-type surfactant into phospholipid vesicle membrane

The incorporation behavior of some anionic surfactants, including amino acid-type surfactants, on phospholipid vesicles was investigated. This was done by measuring the release of a vesicle-entrapped fluorescence probe and the scattered light intensities of vesicle particles in the surfactant solution as a function of surfactant concentration and time. Sodium dodecyl sulfate, sodium dodecanesulfonate, sodium dodecanoyl sarcosinate, and sodium dodecanoyl glutamate were employed in this study. All surfactants ruptured the phospholipid vesicle at around each critical micelle concentration by mixed micelle formation with phospholipid. While leakage of the fluorescence probe took place at a very low concentration in the sulfate- or sulfonate-type surfactant systems, it occurred at the concentration just below the CMC in the amino acid-type surfactant systems. Kinetic analysis of the release of the probe from the vesicles showed that the former surfactants adsorbed independently and homogeneously onto the phospholipid vesicles, while the latter surfactants were cooperatively incorporated.

Kinetics of Factor X activation by the membrane-bound complex of Factor IXa and Factor VIIIa.

Intrinsic tenase consists of activated Factors IX (IXa) and VIII (VIIIa) assembled on a negatively charged phospholipid surface. In vivo, this surface is mainly provided by activated platelets. In vitro, phosphatidylcholine/phosphatidylserine vesicles are often used to mimic natural pro-coagulant membranes. In the present study, we developed a quantitative mathematical model of Factor X activation by intrinsic tenase. We considered two situations, when complex assembly occurs on either the membrane of phospholipid vesicles or the surface of activated platelets. On the basis of existing experimental evidence, the following mechanism for the complex assembly on activated platelets was suggested: (i) Factors IXa, VIIIa and X bind to their specific platelet receptors; (ii) bound factors form complexes on the membrane: platelet-bound Factor VIIIa provides a high-affinity site for Factor X and platelet-bound Factor IXa provides a high-affinity site for Factor VIIIa; (iii) the enzyme-cofactor-substrate complex is assembled. This mechanism allowed the explanation of co-operative effects in the binding of Factors IXa, VIIIa and X to platelets. The model was reduced to obtain a single equation for the Factor X activation rate as a function of concentrations of Factors IXa, VIIIa, X and phospholipids (or platelets). The equation had a Michaelis-Menten form, where apparent V(max) and K(m) were functions of the factors' concentrations and the internal kinetic constants of the system. The equation obtained can be used in both experimental studies of intrinsic tenase and mathematical modelling of the coagulation cascade. The approach of the present study can be applied to research of other membrane-dependent enzymic reactions.

Determination of liposomal encapsulation efficiency using proton NMR spectroscopy

A rapid and simple approach using 1 H NMR was developed for determination of liposomal encapsulation efficiency without the need for physical separation of entrapped and non-entrapped marker. Measurements were made using a marker (homocarnosine) with a pH-sensitive 1 H chemical shift in the presence of a pH gradient across the phospholipid vesicle membrane, or by addition of the chemical shift reagent, thulium(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-(methylene phosphonic acid sodium salt) (TmDOTP 5−). The measured encapsulation efficiencies for the liposomal dispersions prepared from 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) through extrusion using 50, 200 and 1000 nm polycarbonate membranes were found to be identical using the two different experimental approaches.

Orientation and conformation of cell-penetrating peptide penetratin in phospholipid vesicle membranes determined by polarized-light spectroscopy.

The orientation and conformation of the cell-penetrating peptide "penetratin" associated with phospholipid vesicle membranes has been determined using polarized-light spectroscopy. The magnitude of orientation of penetratin is unprecedented for a solute in our membrane system, which we believe indicates a strong, specific interaction with the membrane. To validate the spectroscopic technique for studying the orientation of the two tryptophan residues of penetratin, we applied tryptophan octyl ester as a model compound. It is found to be incorporated in the membrane and preferentially oriented with its hydrophobic benzene edge of the indole chromophore pointing into the membrane and its hydrophilic groups oriented toward the water. For penetratin, the results indicate that a central alpha-helical part of the peptide is aligned parallel with the membrane surface, while the ends of the peptide adopt a planar structure. The planes of the two tryptophan side chains show a preferred orientation parallel with the membrane surface, indicating that they are not inserted into the membrane.

Synergy in K+/H+ exchange across phospholipid vesicle membranes with combinations of valinomycin and chlorophenolsThe T-jump experiments described in this work were carried out during James Hudson's visit to Tata Institute of Fundamental Research, Mumbai 400 005, India during 2000–2001.

The protonophoric mobilities of mixtures of chlorophenols A1 and A2, inferred from a study of the decay of the pH difference across soybean phospholipid vesicular membrane (=ΔpH), have shown that there is a significant synergy for certain combinations of A1 and A2 in the presence of valinomycin (VAL). The results have shown that in addition to a term proportional to [VAL]0[Ai]0, a term proportional to the product of the concentrations [VAL]0[A1]0[A2]0 (with A1 = A2 giving a quadratic term in the “two species case”) contributes to the ΔpH relaxation rate (=1/τ). The magnitudes of these terms depend on the choice of chlorophenols. The former term has been assigned to the well-known mechanism in which the anion A− and the metal ion K+ are back transported across the membrane in the form of the ternary complex VAL–K+–A−. The latter term has been assigned to the less known catalyzed transport mechanism in which A1− is transported from the polar region of the membrane to the non-polar region in the form of the electro-neutral complex VAL–K+–A1− in a fast step followed by translocation through the non-polar region as A1− after dissociation from the ternary complex assisted by a complexing A2− from the polar region. The translocation of VAL–K+ across the membrane facilitates the compensating charge flux of K+ ions in this mechanism. The processes characterized in this work are useful in improving our understanding of the toxicity of uncouplers and uncoupler mixtures and may also help in the development of appropriate analytical tools.

Epoxidation of benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 in reconstituted membranes. Effects of charge and nonbilayer phase propensity of the membrane.

Human cytochrome P4501A1 (CYP1A1) is one of the key enzymes in the bioactivation of environmental pollutants such as benzo[a]pyrene (B[a]P) and other polycyclic aromatic hydrocarbons. To evaluate the effect of membrane properties and distinct phospholipids on the activity of human CYP1A1 purified insect cell-expressed human CYP1A1 and of human NADPH-P450 reductase were reconstituted into phospholipid vesicle membranes. Conversion rates of up to 36 pmol x min(-1) x pmol(-1) CYP1A1 of the enantiomeric promutagens (-)- and (+)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P (7,8-diol) to the genotoxic diolepoxides were achieved. The highest rates were obtained when negatively charged lipids such as phosphatidylserine and phosphatidylinositol and/or nonbilayer phospholipids such as phosphatidylethanolamine were present in the membrane together with neutral lipids. Both Vmax and Km values were changed. This suggests a rather complex mechanism of stimulation which might include altered substrate binding as well as more effective interaction between CYP1A1 and NADPH-P450 reductase. Furthermore, the ratio of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE2) to r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE1) formed from (-)-7,8-diol was significantly increased by the introduction of anionic lipids, but not by that of nonbilayer lipids. Thus, charged lipids affect the stereoselectivity of the epoxidation by leading to the formation of a larger amount of the ultimate mutagen DE2 than of DE1, which is far less carcinogenic. These data suggest that membrane properties such as negative charge and nonbilayer phase propensity are important for the efficiency and selectivity of enzymatic function of human CYP1A1.

Microtubes and nanotubes of a phospholipid bilayer membrane

We propose a theory describing the stable structure of a phospholipid bilayer in pure water involving a spherical mother vesicle with long thin tubular protrusion. It is considered that the phospholipid molecules are in general anisotropic with respect to the axis normal to the membrane and can orient in the plane of the membrane if the curvature field is strongly anisotropic. Taking this into account, the membrane free energy is derived starting from a single-molecule energy and using methods of statistical mechanics. By linking the description on the microscopic level with the continuum theory of elasticity we recover the expression for the membrane bending energy and obtain an additional (deviatoric) contribution due to the orientational ordering of the phospholipid molecules. It is shown that the deviatoric contribution may considerably decrease the phospholipid vesicle membrane free energy if the vesicle involves regions where the difference between the two principal curvatures is large (thin cylindrical protrusions and/or thin finite necks) and thereby yields a possible explanation for the stability of the long thin tubular protrusions of the phospholipid bilayer vesicles. We report on the experiment exhibiting a stable shape of the spherical phospholipid vesicle with a long thin tubular protrusion in pure water.

Liposome technology and the development of Myocet™ (liposomal doxorubicin citrate)

<h2>Summary</h2> Liposomes are phospholipid membrane vesicles that can act as carriers for drugsin order to target them to specific tissues. Encapsulation within a liposome can confer benefits such as longer circulation times, higher stability and reduced toxicity due to targeting of the active drug. Important features that can alter the biodistribution or pharmacokinetics of loaded liposomes include the liposome size, lipid-to-drug ratio, surface characterization (e.g. pegylation) and the method of loading. Myocet was developed in order to target doxorubicin to tumour sites while sparing healthy tissues; in particular the heart. The Myocet loading technique involves a pH gradient and a citrate complex, which allows a very high drug-to-lipid ratioand the use of small liposomes that can accumulate in the tumour through the leaky vasculature that supplies them. Preclinical studies indicate that Myocet accumulation in tumour tissue is more persistent and pervasive than that of conventional doxorubicin. In addition, Myocet is targeted away from the heart, leading to reduced cardiotoxicity without compromising antitumour efficacy.

Electroactive Polymerized Vesicles as Stable Biomaterials for Charge Storage Systems and Electron Coupling Applications

ABSTRACTDiacetylenic phospholipids with disulfide functionality were synthesized and incorporated at dopant levels with unmodified diacetylenic phosphatidylcholine lipids in polymerized vesicles. These polymerized vesicles were immobilized on Au films and structurally characterized in situ using AFM and ESEM and demonstrated surface stability of at least three days. Polymerized diacetylenic phospholipid vesicles displayed retention of entrapped ferricyanide with a half-life of 2.4 weeks at pH 7 and 25 oC. Membrane-bound benzoquinone mediated electron transport across polymerized vesicle membranes providing an initial current of 25 mA and a final charge capacity based on entrapped ferricyanide of 3.5 Ahr/kg. Electron transport across polymerized diacetylenic phospholipid vesicle membranes was described by a pseudosteady-state transport model and was found to be diffusion limited.

Annexin 5 mediates a peroxide-induced Ca2+ influx in B cells

Annexin 5 is a Ca2+-binding protein, the function of which is poorly understood. Structural and electrophysiological studies have shown that annexin 5 can mediate Ca2+ fluxes across phospholipid membranes in vitro[1]. There is, however, no direct evidence for the existence of annexin 5 Ca2+ channels in living cells. Here, we show that annexin 5 inserts into phospholipid vesicle membranes at neutral pH in the presence of peroxide. We then used targeted gene disruption to explore the role of annexin 5 in peroxide-induced Ca2+ signaling in DT40 pre-B cells. DT40 clones lacking annexin 5 exhibited normal Ca2+ responses to both thapsigargin and B-cell receptor stimulation, but lacked the sustained phase of the response to peroxide. This late phase was due to Ca2+ influx from the extracellular space, demonstrating that annexin 5 mediates a peroxide-induced Ca2+ influx. Thus, peroxide induces annexin 5 membrane insertion in vitro, and peroxide-induced Ca2+ entry in vivo in DT40 cells requires annexin 5. Our results are consistent with a role for annexin 5 either as a Ca2+ channel, or as a signaling intermediate in the peroxide-induced Ca2+-influx pathway.

Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy

Interactions of the peptides melittin and magainin with phospholipid vesicle membranes have been studied using fluorescence correlation spectroscopy. Molecular interactions of melittin and magainin with phospholipid membranes are performed in rhodamine-entrapped vesicles (REV) and in rhodamine-labelled phospholipid vesicles (RLV), which did not entrap free rhodamine inside. The results demonstrate that melittin makes channels into vesicle membranes since exposure of melittin to vesicles causes rhodamine release only from REV but not from RLV. It is obvious that rhodamine can not be released from RLV because the inside of RLV is free of dye molecules. In contrast, magainin breaks vesicles since addition of magainin to vesicles results in rhodamine release from both REV and RLV. As the inside of RLV is free of rhodamine, the appearance of rhodamine in solution confirms that these vesicles are broken into rhodamine-labelled phospholipid fragments after addition of magainin. This study is of pharmaceutical significance since it will provide insights that fluorescence correlation spectroscopy can be used as a rapid protocol to test incorporation and release of drugs by vesicles.