Lipid Transition Research Articles

Phase Transition and Crystallization of Lipids

Phase Transition and Crystallization of Lipids

Seasonal changes in minor membrane phospholipid classes, sterols and tocopherols in overwintering insect, Pyrrhocoris apterus

Ectotherm animals including insects are known to undergo seasonal restructuring of the cell membranes in order to keep their functionality and/or protect their structural integrity at low body temperatures. Studies on insects so far focused either on fatty acids or on composition of molecular species in major phospholipid classes. Here we extend the scope of analysis and bring results on seasonal changes in minor phospholipid classes, lysophospholipids (LPLs), free fatty acids, phytosterols and tocopherols in heteropteran insect, Pyrrhocoris apterus. We found that muscle tissue contains unusually high amounts of LPLs. Muscle and fat body tissues also contain high amounts of β-sitosterol and campesterol, two phytosterols derived from plant food, while only small amounts of cholesterol are present. In addition, two isomers (γ and δ) of tocopherol (vitamin E) are present in quantities comparable to, or even higher than phytosterols in both tissues. Distinct seasonal patterns of sterol and tocopherol concentrations were observed showing a minimum in reproductively active bugs in summer and a maximum in diapausing, cold-acclimated bugs in winter. Possible adaptive meanings of such changes are discussed including: preventing the unregulated transition of membrane lipids from functional liquid crystalline phase to non-functional gel phase; decreasing the rates of ion/solute leakage; silencing the activities of membrane bound enzymes and receptors; and counteracting the higher risk of oxidative damage to PUFA in winter membranes.

Connected and isolated CH2 populations in acyl chains and its relation to pockets of confined water in lipid membranes as observed by FTIR spectrometry

Analysis of the band corresponding to the frequency of vibrational symmetric stretching mode of methylene groups in the lipid acyl chains and the bands of water below and above the phase transition of different lipids by Fourier transform infrared spectroscopy gives strong support to the formation of confined water pockets in between the lipid acyl chains. Our measures and analysis consolidate the mechanism early proposed by Traüble, in the sense that water is present in kinks formed by trans-gauche isomers along the hydrocarbon tails. The formation of these regions depends on the acyl lipid composition, which determines the presence of different populations of water species, characterized by its degree of H bond coordination in fluid saturated or unsaturated lipids. The free energy excess due to the reinforcement of the water structure along few water molecules in the adjacencies of exposed membrane residues near the phase transition is a reasonable base to explain the insertion and translocation of polar peptides and aminoacid residues through the biomembrane on thermodynamic and structural grounds.

Water Pockets between Acyl Chains and its Relation to Peptide Insertion in Lipid Membranes

Presence of water in the membrane structure has been invoked to interpret the insertion of highly hydrophilic aminoacid moieties in pools between acyl chains, described as water pockets. Analysis of the band corresponding to the frequency of vibrational symmetric stretching mode of methylenes and the bands of water below and above the phase transition of different lipids by Fourier transform infrared spectroscopy give strong support to the formation of confined water pockets in between the lipid acyl chains. We present a rational description of the water pockets creation and the influence of the adjacent wall formed at the phase transition by analyzing the changes of FTIR-ATR spectra in the regions corresponding to the CH2 and water bands shifts with temperature. The presence of water in these packing defects gives support to the creation of regions with free energy excess due to reinforcement of the water structure. This high energy defects explains the insertion of peptides and aminoacid residues and translocation of peptides through the biomembrane.

Interactions of a Monofluorinated Phospholipid with Saturated Phosphatidylcholines of Different Chain Lengths

This study uses differential scanning calorimetry (DSC) and fluorescence spectroscopy to study the thermodynamic and kinetic effects of the monofluorinated phospholipid 1-palmitoyl-2-[16-fluoropalmitoyl]-phosphatidylcholine (F-DPPC) on bilayers composed of the fully saturated phosphatidylcholines 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In both bilayer systems, DSC thermograms indicate a disappearance of pretransition peaks (Tp) along with a rise in main transition (Tm) hysteresis at elevated F-DPPC mol%. Fluorescence intensity measurements reveal an inverse relationship between F-DPPC mol% and the emission intensity of the environment-sensitive probe 1,6-diphenyl-1,3,5-hexatriene (DPH) below the main transitions of the respective lipids. These trends suggest a growth in interdigitated domains with the incorporation of additional F-DPPC into the bilayer. Significant drops in intensity values were observed at lower F-DPPC mole percentages in the DSPC system than the DMPC system, indicating that the latter lipid has a higher threshold for F-DPPC-induced interdigitation. Our results support that F-DPPC encourages the interdigitated phase (LβI) in saturated bilayers and highlight the stabilizing effect that long acyl chains have on this phase.

Materials characterization of the low temperature sensitive liposome (LTSL): effects of the lipid composition (lysolipid and DSPE–PEG2000) on the thermal transition and release of doxorubicin

This paper describes how we have used material science, physical chemistry, and some luck, to design a new thermal-sensitive liposome (the low temperature sensitive liposome (LTSL)) that responds at clinically attainable hyperthermic temperatures releasing its drug in a matter of seconds as it passes through the microvasculature of a warmed tumor. The LTSL is composed of a judicial combination of three component lipids, each with a specific function and each affecting specific material properties, including a sharp thermal transition and a rapid on-set of membrane permeability to small ions, drugs and small dextran polymers. Experimentally, the paper describes how bilayer-concentration changes involving the lysolipid and the presence or absence of DSPE-PEG2000 affect both the lipid transition temperature and the drug release. While the inclusion of 4 mol% DSPE-PEG2000 raises the transition temperature peak (T(m)) by about 1 degrees C, the inclusion of 5.0, 9.7, 12.7 and 18.0 mol% MSPC slightly lowered this peak back to 41.7 degrees C, while not further broadening the peak breadth. As for drug release, in the absence of MSPC, the encapsulated doxorubicin-citrate is hardly released at all. Increasing the composition of MSPC in the lipid mixture (5.0, 7.4, 8.5 and 9.3 mol% MSPC) shows faster and faster initial doxorubicin release rates, with 8.5 and 9.3 mol% MSPC formulations giving 80% of encapsulated drug released in 4 and 3 min, respectively. The Thermodox formulation (9.7 mol% MSPC) gives 60% released in the first 20 s. The presence of PEG-lipid is found to be essential in order for the lysolipid-induced permeability to reach these very fast times. From drug and dextran release experiments, and estimates of the molecular and pore size, the conclusions are that: in order to induce lasting nanopores in lipid bilayers -10 nm diameter, they initially require the presence (from the solid phase structure) of grain boundary defects at the DPPC transition and the permeabilizing component(s) can either be a pore forming lysolipid/surfactant plus a PEG-lipid, or can be generated by a PEG-surfactant incorporated at -4-5 mol%. The final discussion is centered around the postulated defect structures that result in membrane leakage and the permeability of doxorubicin and H+ ions.

A study on the interaction of nanoparticles with lipid membranes and their influence on membrane fluidity

In recent years, liposomes encapsulated with nanoparticles have found enormous scopes in various biomedical fields such as drug design, transport, imaging, targeted delivery and therapy. These applications require a clear understanding about the interaction of nanoparticles with cell membranes. The present work aims to investigate the effect of encapsulation of uncharged and positively charged nanoparticles in three different types of lipids such as1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine(SOPC-POPS) mixture and archaeal lipids. Through the temperature dependent fluorescence anisotropy measurements, we have found that the entrapment of nanoparticles in the bilayer has decreased the lipid transition temperature and increased the membrane fluidity of all three types of lipid vesicles. The results were more predominant in SOPC-POPS mixture because of high density encapsulation of nanoparticles in the vesicles due to electrostatic interaction between negatively charged membrane and positively charged iron oxide nanoparticles.

The potential of scanning microcalorimetry for studying thermotropic conformational transitions in biomacromolecules1

General operational principles of a scanning microcalorimeter, methods and approaches to obtaining thermodynamic and kinetic information on the studied transition, and the existence of correlations between the structural features of a macromolecule and the measured thermodynamic parameters of denaturation transitions are considered. The results of studying the domain organization for a number of proteins and transfer poly(ribonucleic acids), the kinetics of heat denaturation, and the nature of the transitional (activated) state of macromolecules are surveyed. The potential of high-pressure scanning microcalorimetry for measuring volume changes during conformational transitions of proteins and lipids is discussed.

Lipid Bilayers and Membrane Dynamics: Insight into Thickness Fluctuations

Thickness fluctuations have long been predicted in biological membranes but never directly observed experimentally. Here, we utilize neutron spin echo spectroscopy to experimentally reveal such fluctuations in a pure, fully saturated, phosphocholine lipid bilayer system. These fluctuations appear as an excess in the dynamics of undulation fluctuations. Like the bending rigidity, the thickness fluctuations change dramatically as the lipid transition temperature is crossed, appearing to be completely suppressed below the transition. Above the transition, the relaxation rate is on the order of 100 ns and is independent of temperature. The amplitude of the thickness fluctuations is 3.7 Å ± 0.7 Å, which agrees well with theoretical calculations and molecular dynamics simulations. The dependence of the fluctuations on lipid tail lengths is also investigated and determined to be minimal in the range of 14 to 18 carbon tails.

The physical properties of lipids and their role in controlling the distribution of zooplankton in the oceans

A new perspective on the role of lipids in zooplankton is proposed, with solid – liquid phase transitions of lipids being a factor regulating their buoyancy. These phase transitions are controlled by zooplankton in relation to their physical environment, through the selective accumulation of specific lipids with optimum levels of unsaturation. The necessity to control buoyancy and maintain an optimum depth is a fundamental evolutionary force, driving anatomical, biochemical and behavioural adaptations of all organisms within the aquatic realm. It is hypothesized that each species adjusts the amount, composition and anatomical location of lipids, to maximize fitness according to their preferred habitat and life history traits. Recent discoveries regarding the role of phase transitions of lipids in marine zooplankton and their role in regulating buoyancy will require re-interpretation of existing data and stimulate future scientific endeavours in zooplankton research.

Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while (31) P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization.

Light scattering measurement and Avrami analysis of the lamellar to inverse hexagonal phase transition kinetics of the lipid DEPE

The lamellar to inverse hexagonal phase transition of lipids is much studied as a model for understanding cellular processes such as membrane fusion and pore formation. Much remains unknown, including a theoretical understanding and a definitive value of the phase transition temperature for DEPE, as literature values vary over 10°C. Avrami theory has been commonly used to analyze phase transition kinetics. However, to the best of our knowledge, Avrami theory has not been used to analyze the lamellar to inverse hexagonal transition in lipids until now. We used laser light scattering to measure phase transition temperature of the lipid DEPE (1,2-dielaidoyl-sn-phosphatidylethanolamine) and found it to be 61.0±0.5°C. We found the hysteresis, |Tmeasured−Tequilibrium|, scaled as rβ, where r is the ramp rate and β=0.29±0.02. This is the same power law behavior found by others for an isomer of DEPE known as DOPE (1,2-dioleoyl-sn-glycero-3 ethanolamine); however, DEPE exhibits roughly half the hysteresis of DOPE. An analysis of DEPE kinetics yields Avrami exponents ranging from 1 to 7, suggesting the transition propagates one dimensionally and is initiated by a widely varying nucleation rate.

Penetratin-induced transdermal delivery from HII mesophases of sodium diclofenac

Penetratin, a cell penetrating peptide is embedded within a reversed hexagonal (HII) mesophase for improved transdermal delivery of sodium diclofenac (Na-DFC). The HII mesophase serves as the solubilization reservoir and gel matrix whereas penetratin is the transdermal penetration enhancer for the drug. The systems were characterized and the interactions between the components were determined by SAXS, ATR-FTIR and SD-NMR. High affinity of Na-DFC to glycerol monooleate (GMO) was revealed, associated with increasing the order within the water channels. This affinity is enhanced upon heating and seems to be associated with GMO dehydration. Penetratin (PEN) is entrapped at the hydrophilic region of the HII mesophase, between the GMO headgroups, reducing the order of the system and decreasing the size of the hexagonal domains.The transdermal delivery rate of Na-DFC through porcine skin, from the HII mesophases, was enhanced by PEN and so also the cumulative transport crossing the skin. PEN induced accelerated drug diffusion through the stratum corneum, towards the different skin layers.The transdermal delivery enhancement is explained from the results of the ATR-FTIR analysis. It seems that PEN accelerates the structural transition of skin lipids from hexagonal to liquid. The disordering results in enhanced diffusion of Na-DFC through the stratum corneum, followed by enhanced overall penetration of the drug.

A universal method for planar lipid bilayer formation by freeze and thaw

A procedure based on freezing and thawing was developed to induce the rupture of adsorbed lipid vesicles on solid surfaces into supported lipid bilayers (SLBs). The SLB assembly exploits the phase transition of both lipids and water during freezing. It enables SLB formation independent of the type of substrates and lipids as long as the vesicles spontaneously adsorb onto the surface. The created SLB is a single bilayer, and has a diffusion coefficient of (0.6–4) × 10−8 cm2 s−1 on TiO2, which is in the same range as the SLBs formed by conventional techniques. The presented approach has the advantages of both the Langmuir–Blodgett method (the versatility in the selection of lipids and substrates) and vesicle fusion (self-assembly) at the same time.

Structural Plasticity in Self-Assembling Transmembrane β-Sheets

Here we test the hypothesis that membrane-spanning β-sheets can exhibit structural plasticity in membranes due to their ability to shift hydrogen-bonding patterns. Transmembrane β-sheet forming peptides of the sequence AcWLn, where n = 5, 6, or 7, which range from 21 to 27 Å in maximum length, were incorporated into bilayers made of phosphatidylcholine lipids with saturated acyl chains containing 14, 16, or 18 carbons, which are 36–50 Å in thickness. The effect of the peptide β-sheets on fluid- and gel-phase bilayers were studied with differential scanning calorimetry and circular dichroism spectroscopy. We show that AcWL5 forms a stable, peptide-rich gel phase in all three lipids. The whole family of AcWLn peptides appears to form similarly stable, nonmembrane-disrupting β-sheets in all bilayer phases and thicknesses. Bilayers containing up to 20 mol % peptide, which is the maximum concentration tested, formed gel phases with melting temperatures that were equal to, or slightly higher than, the pure lipid transitions. Given the range of peptide lengths and bilayer thicknesses tested, these experiments show that the AcWLn family of membrane-inserted β-sheets exhibit remarkable structural plasticity in membranes.

Partial dehydration and cryopreservation of Citrus seeds

Three categories of seed storage behavior are generally recognized among plant species: orthodox, intermediate and recalcitrant. Intermediate seeds cannot be stored in liquid nitrogen (LN) without a previous partial dehydration process. The water content (WC) of the seeds at the moment of immersion in LN must be regarded as the most critical factor in cryopreservation. The purpose of this study was to investigate the basis of the optimal hydration status for cryopreservation of Citrus seeds: C. sinensis (sweet orange), C. paradisi (grapefruit), C. reticulata (mandarin) in LN. To study the tolerance to dehydration and LN exposure, seeds were desiccated by equilibration at relative humidities between 11 and 95%. Sorption isotherms were determined and modeled; lipid content of the seeds was measured. Seed desiccation sensitivity was quantified by the quantal response model. Differential scanning calorimetry (DSC) thermograms were determined on cotyledon tissue at different moisture contents to measure ice melting enthalpies and unfrozen WC. Samples of total seed lipid extract were also analyzed by DSC to identify lipid transitions in the thermograms. The limit of hydration for LN Citrus seeds treatment corresponded to the unfrozen WC in the tissue, confirming that seed survival strictly depended on avoidance of intracellular ice formation.

The Physics of Nerves and Lipid Membrane Channels

At physiological temperature, many biomembranes are found in a physical state slightly above the melting transition of the membrane lipids. We show that this condition gives rise to the possibility of the propagation of electromechanical pulses (solitons) in membrane cylinders that share many properties with the action potential in nerves. Among those properties are the reversible heat change measured in nerves under the influence of the action potential, a mechanical shortening and thickening of the nerve, but also the excitability by voltage, local cooling, and by mechanical stimulus. Depending on boundary conditions one can obtain voltage pulse trains with minimum pulse distance (refractory periods) and undershoot (hyperpolarization).The underlying physics is that of the fluctuation-dissipation theorem, that contains strict thermodynamic couplings between heat capacity, compressibility and lifetimes of membrane processes. It contains a role for all thermodynamic variables (not only voltage). We show that our electromechanical picture also contains a mechanism for anesthesia that lies in the melting point depression caused by these drugs, in agreement with the well-known Meyer-Overton correlation for anesthetics. The presence of anesthetics results in the reduction of the membrane excitability. Further, a direct consequence of the membrane fluctuations is the observation of spontaneous formation of ion-channel-like conduction events in the pure lipid membrane indistinguishable from those attributed to ion channel proteins. Since the theory is of macroscopic thermodynamic nature it does not make statements on processes on the molecular scale. However, it is consistent with known pharmacology provided that the macroscopic conservation of heat is maintained.Summarizing, we provide thermodynamic picture for many excitatory processes in biomembranes that let pulse propagation, ion conductance, and action of drugs all be part of one self-consistent physical description.

Volume and Expansivity Changes of Micelle Formation Measured by Pressure Perturbation Calorimetry

We present the application of pressure perturbation calorimetry (PPC) as a new method for the volumetric characterization of the micelle formation of surfactants. The evaluation is realized by a global fit of PPC curves at different surfactant concentration ranging, if possible, from below to far above the CMC. It is based on the knowledge of the temperature dependence of the CMC, which can for example be characterized by isothermal titration calorimetry. We demonstrate the new approach for decyl-β-maltopyranoside (DM). It shows a strong volume increase upon micelle formation of 16 ± 2.5 mL/mol (+4%) at 25 °C, and changes with temperature by -0.1 mL/(mol K). The apparent molar expansivity (E(S)) decreases upon micelle formation from 0.44 to 0.31 mL/(mol K) at 25 °C. Surprisingly, the temperature dependence of the expansivity of DM in solution (as compared with that of maltose) does not agree with the principal behavior described for polar (E(S)(T) decreasing) and hydrophobic (E(S)(T) increasing) solutes or moieties before. The results are discussed in terms of changes in hydration of the molecules and internal packing of the micelles and compared with the volumetric effects of transitions of proteins, DNA, lipids, and polymers.

Self-Consistent Field Modeling of Three-Dimensional Morphologies of Branched Lipid Surfactant at Air-Water Interface

Three-dimensional mesoscopic morphologies and the thermodynamics of structural phase transitions of amphiphilic lipids at air-water interfaces are studied using self-consistent field theory. Changing the relative amount of lipids in the system led to a series of 3D morphologic phases with varying average interfacial area per molecule, mimicking a compression of the model membranes. Membranes of both saturated and unsaturated lipids undergo a transition from cylindrical micelle to lamella when the lipid content in the system increases from 2% to about 19–20%. With further increase in the lipid content, saturated lipids first develop non-uniform quasi-2D distributions in the lamella and then gradually transform into a hybrid morphology containing quasi-planar lamellae. In contrast, unsaturated lipids develop reverse-micellar morphologies.

Effect of pressure on the bilayer phase transitions of asymmetric lipids with an unsaturated acyl chain in sn-1 position

The bilayer phase transitions of a series of mixed-chain phosphatidylcholines (PCs) with an unsaturated chain in the sn-1 position and saturated chain with different lengths in the sn-2 position, 1-oleoyl-2-myristoyl-PC (OMPC), 1-oleoyl-2-palmitoyl-PC (OPPC) and 1-oleoyl-2-stearoyl-PC (OSPC), were observed by differential scanning calorimetry (DSC) under ambient pressure and light transmittance measurements under high pressure. All the PC bilayer membranes exhibited not only the main transition from the lamellar gel (Lβ) phase to the liquid crystalline (Lα) phase but also the transition from the lamellar crystal (Lc) to the Lβ (or Lα) phase. The thermodynamic quantities of the transition between the Lc and Lα phases for bilayers of unsaturated mixed-chain PCs were compared with those of the corresponding mixed-chain PCs with saturated acyl chains such as 1-stearoyl-2-myristoyl-PC (SMPC), 1-stearoyl-2-palmitoyl-PC (SPPC) and 1,2-distearoyl-PC (DSPC). It turned out that the partial molar quantities of the unsaturated mixed-chain PC bilayers in the Lc phase are larger than those of the saturated mixed-chain PCs bilayers. The increase in the partial molar quantities in the Lc phase by the introduction of a cis double bond into the sn-1 acyl chain is attributable to the loose packing of the saturated and unsaturated acyl chains in the Lc phase.