Lipid Phase State Research Articles

Electric Fields at the Lipid Membrane Interface.

This review presents a comprehensive analysis of electric field distribution at the water-lipid membrane interface in the context of its relationship to various biochemical problems. The main attention is paid to the methodological aspects of bioelectrochemical techniques and quantitative analysis of electrical phenomena caused by the ionization and hydration of the membrane-water interface associated with the phase state of lipids. One of the objectives is to show the unique possibility of controlling changes in the structure of the lipid bilayer initiated by various membrane-active agents that results in electrostatic phenomena at the surface of lipid models of biomembranes-liposomes, planar lipid bilayer membranes (BLMs) and monolayers. A set of complicated experimental facts revealed in different years is analyzed here in order of increasing complexity: from the adsorption of biologically significant inorganic ions and phase rearrangements in the presence of multivalent cations to the adsorption and incorporation of pharmacologically significant compounds into the lipid bilayer, and formation of the layers of macromolecules of different types.

A new look at Hsp70 activity in phosphatidylserine-enriched membranes: chaperone-induced quasi-interdigitated lipid phase

70 kDa heat shock protein Hsp70 (also termed HSP70A1A) is the major stress-inducible member of the HSP70 chaperone family, which is present on the plasma membranes of various tumor cells, but not on the membranes of the corresponding normal cells. The exact mechanisms of Hsp70 anchoring in the membrane and its membrane-related functions are still under debate, since the protein does not contain consensus signal sequence responsible for translocation from the cytosol to the lipid bilayer. The present study was focused on the analysis of the interaction of recombinant human Hsp70 with the model phospholipid membranes. We have confirmed that Hsp70 has strong specificity toward membranes composed of negatively charged phosphatidylserine (PS), compared to neutral phosphatidylcholine membranes. Using differential scanning calorimetry, we have shown for the first time that Hsp70 affects the thermotropic behavior of saturated PS and leads to the interdigitation that controls membrane thickness and rigidity. Hsp70-PS interaction depended on the lipid phase state; the protein stabilized ordered domains enriched with high-melting PS, increasing their area, probably due to formation of quasi-interdigitated phase. Moreover, the ability of Hsp70 to form ion-permeable pores in PS membranes may also be determined by the bilayer thickness. These observations contribute to a better understanding of Hsp70-PS interaction and biological functions of membrane-bound Hsp70 in cancer cells.

Interfacial Aβ fibril formation is modulated by the disorder-order state of the lipids: The concept of the physical environment as amyloid inductor in biomembranes

The behavior of amphiphilic molecules such as lipids, peptides and their mixtures at the air/water interface allow us to evaluate and visualize the arrangement formed in a confined and controlled surface area. We have studied the surface properties of the zwitterionic DPPC lipid and Aβ(1–40) amyloid peptide in mixed films at different temperatures (from 15 to 40 °C). In this range of temperature the surface properties of pure Aβ(1–40) peptide remained unchanged, whereas DPPC undergoes its characteristic liquid-expanded → liquid-condensed bidimensional phase transition that depends on the temperature and lateral pressure. This particular property of DPPC makes it possible to dynamically study the influence of the lipid phase state on amyloid structure formation at the interface in a continuous, isothermal and abrupt change on the environmental condition. As the mixed film is compressed the fibril-like structure of Aβ(1–40) is triggered specifically in the liquid-expanded region, independently of temperature, and it is selectively excluded from the well-visible liquid condensed domains of DPPC. The Aβ amyloid fibers were visualized by using BAM and AFM and they were Thio T positive. In mixed DPPC/Aβ(1–40) films the condensed domains (in between 11 mN/m to 20 mN/m) become irregular probably due to the fibril-like structures is imposing additional lateral stress sequestering lipid molecules in the surrounding liquid-expanded phase to self-organize into amyloids.

Impact of the lipid phase composition and state on the in vitro digestibility and chlorophyllin bioaccessibility of W1/O/W2 emulsions into whole milk

Water-in-oil-in-water (W1/O/W2) emulsions offer the potential to deliver hydrophilic bioactive compounds into foods, yet their application remains limited due to their instability. Thus, the impact of lipid phase composition and state on the colloidal stability, in vitro lipid digestibility and chlorophyllin (CHL) bioaccessibility of W1/O/W2 emulsions before and after incorporation into whole milk was studied. Medium-chain triglyceride oil (MCT) was used as a liquid lipid phase and MCT with glyceryl stearate (GS) or pure hydrogenated palm oil (HPO) as gelled lipid phases. The lipid phase composition was crucial to forming W1/O/W2 emulsions. MCT or MCT+GS allowed the successful formation of W1/O/W2 emulsions, being more stable upon gastric conditions those formulated with MCT+GS than pure MCT. In contrast, the use of HPO led to phase separation, which was maintained after the gastric conditions. Regarding their lipid digestibility, W1/O/W2 emulsions formulated with MCT or MCT+GS were fully digested, whereas only 40% of the lipid was digested using HPO. In accordance, the CHL bioaccessibility was higher using MCT or MCT+GS than HPO. When co-digested with whole milk, the colloidal stability and lipid digestibility of the W1/O/W2 emulsions with MCT or MCT+GS were not altered, whereas the W1/O/W2 emulsion-HPO showed enhanced colloidal stability and lipid digestibility (57.71 ± 3.06%), due to the surface-active properties of milk protein. The present study provides useful information to develop stable functional foods enriched with hydrophilic bioactive compounds by using W1/O/W2 emulsions.

Interactions between DMPC Model Membranes, the Drug Naproxen, and the Saponin β-Aescin.

In this study, the interplay among the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) as a model membrane, the nonsteroidal anti-inflammatory drug naproxen, and the saponin β-aescin are investigated. The naproxen amount was fixed to 10 mol%, and the saponin amount varies from 0.0 to 1.0 mol%. Both substances are common ingredients in pharmaceutics; therefore, it is important to obtain deeper knowledge of their impact on lipid membranes. The size and properties of the DMPC model membrane upon naproxen and aescin addition were characterized with differential scanning calorimetry (DSC), small- and wide-angle X-ray scattering (SAXS, WAXS), and photon correlation spectroscopy (PCS) in a temperature-dependent study. The interaction of all substances was dependent on the lipid phase state, which itself depends on the lipid's main phase transition temperature Tm. The incorporation of naproxen and aescin distorted the lipid membrane structure and lowers Tm. Below Tm, the DMPC-naproxen-aescin mixtures showed a vesicle structure, and the insertion of naproxen and aescin influenced neither the lipid chain-chain correlation distance nor the membrane thickness. Above Tm, the insertion of both molecules instead induced the formation of correlated bilayers and a decrease in the chain-chain correlation distance. The presented data clearly confirm the interaction of naproxen and aescin with DMPC model membranes. Moreover, the incorporation of both additives into the model membranes is evidenced.

Improved accuracy and reproducibility of spontaneous liposome leakage measurements by the use of supported lipid bilayer-modified quartz cuvettes

Recent studies have revealed avid interactions between liposomes and several solid materials, such as quartz, polystyrene (PS) and poly(methyl methacrylate) (PMMA), commonly found in cuvettes used for spectroscopic measurements. These interactions risk leading to detrimental changes in liposome structure and integrity that, if overlooked, may compromise the measurements. In case of leakage experiments based on probing the spontaneous release of encapsulated hydrophilic markers, the liposome-cuvette interactions may result in the recording of erroneously high degrees of leakage. In the present study we investigate the possibilities of preventing unwanted liposome-cuvette interactions through the use of quartz cuvettes passivated with supported lipid bilayers (SLBs). The results show that this strategy leads to higher reproducibility and significantly improved accuracy of the leakage measurements. The usefulness of the method is validated in comparative experiments focused on how changes in temperature and lipid phase state, as well as inclusion of poly(ethylene glycol)-conjugated lipids (PEG-lipids), affect the release of liposome encapsulated carboxyfluorescein (CF).

Mechanistic Pathway of Lipid Phase-Dependent Lipid Corona Formation on Phenylalanine-Functionalized Gold Nanoparticles: A Combined Experimental and Molecular Dynamics Simulation Study.

In recent years, the underlying mechanism of formation of the lipid corona and its stability have begun to garner interest in the nanoscience community. However, until now, very little is known about the role of different properties of nanoparticles (NPs) (surface charge density, hydrophobicity, and size) in lipid corona formation. Apart from the physicochemical properties of NPs, the different properties of lipids remain elusive in lipid corona formation. In the present contribution, we have investigated the interaction of phenylalanine-functionalized gold NPs (Au-Phe NPs) with different zwitterionic lipid vesicles of different phase states (sol-gel and liquid crystalline at room temperature) as a function of lipid concentration. The main objective of the present work is to understand how the lipid phase affects lipid corona formation and lipid-induced aggregation in various media. Our results establish that the lipid phase state, area per lipid head group, and the buffer medium play important roles in lipid-induced aggregation. The lipid corona occurs for NPs at high lipid concentration, irrespective of the phase states and area per lipid head group of the lipid bilayer. Notably, the lipid corona also forms at a low concentration of lipid vesicles in the liquid crystalline phase (1,2-dioleoyl-sn-glycero-3-phosphocholine). The corona formation brings in remarkable stability to NPs against freeze-thaw cycles. Based on the stability, for the first time, we classify lipid corona as "hard lipid corona" and "soft lipid corona". This distinct classification will help to develop suitable nanomaterials for various biomedical applications.

Insight into the Exosomal Membrane: From Viewpoints of Membrane Fluidity and Polarity.

Numerous research studies have been done for exosomes, particularly focusing on membrane proteins and included nucleic acids, and the volume of the knowledge about the lipids in the exosomal membrane has been increasing. However, the dynamic property of the exosomal membrane is hardly studied. By employing milk exosome as an example, herein the exosomal membrane was characterized focusing on the membrane fluidity and polarity. The lipid composition and phase state of milk exosome (exosome from bovine milk) were estimated. The milk exosome contained enriched Chol (43.6 mol % in total lipid extracts), which made the membrane in the liquid-ordered (lo) phase by interacting with phospholipids. To suggest a model of exosomal vesicle cargo, the liposome compositions that mimic milk exosome were studied: liposomes were made of cholesterol (Chol), milk sphingomyelin (milk SM), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). By using fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 6-dodecanoyl-2-dimethylaminonaphthalene, the microenvironments of submicron-sized membranes of exosome and model liposomes were investigated. The membrane fluidity of milk exosome was slightly higher than those of Chol/milk SM/POPC liposomes with a similar content of Chol, suggesting the presence of enriched unsaturated lipids. The most purposeful membrane property was obtained by the liposome composition of Chol/milk SM/POPC = 40/15/45. From the above, it is concluded that Chol is a fundamental component of the milk exosomal membrane to construct the enriched lo phase, which could increase the membrane rigidity and contribute to the function of exosome.

Formation and Stabilization of W1/O/W2 Emulsions with Gelled Lipid Phases.

Water-in-oil-in-water (W1/O/W2) emulsions are emulsion-based systems where the dispersed phase is an emulsion itself, offering great potential for the encapsulation of hydrophilic bioactive compounds. However, their formation and stabilization is still a challenge mainly due to water migration, which could be reduced by lipid phase gelation. This study aimed to assess the impact of lipid phase state being liquid or gelled using glyceryl stearate (GS) at 1% (w/w) as well as the hydrophilic emulsifier (T80: Tween 80 or lecithin) and the oil type (MCT:medium chain triglyceride or corn oil (CO) as long chain triglyceride) on the formation and stabilization of chlorophyllin W1/O/W2 emulsions. Their colloidal stability against temperature and light exposure conditions was evaluated. Gelling both lipid phases (MCT and CO) rendered smaller W1 droplets during the first emulsification step, followed by formation of W1/O/W2 emulsions with smaller W1/O droplet size and more stable against clarification. The stability of W1/O/W2 emulsions was sensitive to a temperature increase, which might be related to the lower gelling degree of the lipid phase at higher temperatures. This study provides valuable insight for the formation and stabilization of W1/O/W2 emulsions with gelled lipid phases as delivery systems of hydrophilic bioactive compounds under common food storage conditions.

In Situ Monitoring of Lipid Phase State Make Target Lipid Mixtures Similar to Intercellular Lipid in the Stratum Corneum

AbstractIn this study, lipid structural change is monitored using Raman spectroscopy during heat treatment, along with the impact of lipid states on the structural and physical properties during the preparation process of the dried and hydrated lipid mixture (LM) similar to intercellular lipid in stratum corneum. The microstructures and thermal behavior of these LMs change depending on the melting of lipid ingredients in the preparation process. It is recognized that variable temperature Raman spectroscopy (VT‐Raman) is a useful and attractive tool for the sensitive in situ monitoring of lipid state changes and lipid melting. The LMs can incorporate D2O into their structures regardless of preparation temperature due to increasing lattice distance by hydration. These results suggest that monitoring lipid structural changes during the heating step is important to precisely prepare target LMs.Practical Applications: This study reveals that VT‐Raman is a useful and attractive tool in in situ monitoring of lipid state change and lipid melting. The monitoring of the preparation process by VT‐Raman is necessary to precisely prepare the target LM similar to intercellular lipid of stratum corneum because the microstructures and thermal properties of these LMs change depending on the melting of lipid ingredients during the preparation process.

The Biosurfactant β-Aescin: A Review on the Physico-Chemical Properties and Its Interaction with Lipid Model Membranes and Langmuir Monolayers.

This review discusses recent progress in physicochemical understanding of the action of the saponin -aescin (also called -escin), the biologically active component in the seeds of the horse chestnut tree Aesculus hippocastanum. -Aescin is used in pharmacological and cosmetic applications showing strong surface activity. In this review, we outline the most important findings describing the behavior of -aescin in solution (e.g., critical micelle concentration () and micelle shape) and special physicochemical properties of adsorbed -aescin monolayers at the air–water and oil–water interface. Such monolayers were found to posses very special viscoelastic properties. The presentation of the experimental findings is complemented by discussing recent molecular dynamics simulations. These simulations do not only quantify the predominant interactions in adsorbed monolayers but also highlight the different behavior of neutral and ionized -aescin molecules. The review concludes on the interaction of -aescin with phospholipid model membranes in the form of bilayers and Langmuir monolayers. The interaction of -aescin with lipid bilayers was found to strongly depend on its . At concentrations below the , membrane parameters are modified whereas above the , complete solubilization of the bilayers occurs, depending on lipid phase state and concentration. In the presence of gel-phase phospholipids, discoidal bicelles form; these are tunable in size by composition. The phase behavior of -aescin with lipid membranes can also be modified by addition of other molecules such as cholesterol or drug molecules. The lipid phase state also determines the penetration rate of -aescin molecules into lipid monolayers. The strongest interaction was always found in the presence of gel-phase phospholipid molecules.

Gel Phase 1,2-Distearoyl-sn-glycero-3-phosphocholine-Based Liposomes Are Superior to Fluid Phase Liposomes at Augmenting Both Antigen Presentation on Major Histocompatibility Complex Class II and Costimulatory Molecule Display by Dendritic Cells in Vitro.

Lipid-based nanoparticles have in recent years attracted increasing attention as pharmaceutical carriers. In particular, reports of them having inherent adjuvant properties combined with their ability to protect antigen from degradation make them suitable as vaccine vectors. However, the physicochemical profile of an ideal nanoparticle for vaccine delivery is still poorly defined. Here, we used an in vitro dendritic cell assay to assess the immunogenicity of a variety of liposome formulations as vaccine carriers and adjuvants. Using flow cytometry, we investigated liposome-assisted antigen presentation as well as the expression of relevant costimulatory molecules on the cell surface. Cytokine secretion was further evaluated with an enzyme-linked immunosorbent assay (ELISA). We show that liposomes can successfully enhance antigen presentation and maturation of dendritic cells, as compared to vaccine fusion protein (CTA1-3Eα-DD) administered alone. In particular, the lipid phase state of the membrane was found to greatly influence the vaccine antigen processing by dendritic cells. As compared to their fluid phase counterparts, gel phase liposomes were more efficient at improving antigen presentation. They were also superior at upregulating the costimulatory molecules CD80 and CD86 as well as increasing the release of the cytokines IL-6 and IL-1β. Taken together, we demonstrate that gel phase liposomes, while nonimmunogenic on their own, significantly enhance the antigen-presenting ability of dendritic cells and appear to be a promising way forward to improve vaccine immunogenicity.

Raman study of lipid phase state in freezing mammalian embryos and oocytes

Raman study of lipid phase state in freezing mammalian embryos and oocytes

Lipid Droplet Phase Transition in Freezing Cat Embryos and Oocytes Probed by Raman Spectroscopy

Embryo and oocyte cryopreservation is a widely used technology for cryopreservation of genetic resources. One limitation of cryopreservation is the low tolerance to freezing observed for oocytes and embryos rich in lipid droplets. We apply Raman spectroscopy to investigate freezing of lipid droplets inside cumulus-oocyte complexes, mature oocytes, and early embryos of a domestic cat. Raman spectroscopy allows one to characterize the degree of lipid unsaturation, the lipid phase transition from the liquid-like disordered to solid-like ordered state, and the triglyceride polymorphic state. For all cells examined, the average degree of lipid unsaturation is estimated as ∼1.3 (with ±20% deviation) double bonds per acyl chain. The onset of the lipid phase transition occurs in a temperature range from −10 to +4°C and does not depend on the cell type. Lipid droplets in cumulus-oocyte complexes are found to undergo abrupt lipid crystallization shifted in temperature from the ordering of the lipid conformational state. In the case of mature oocytes and early embryos obtained in vitro, the lipid crystallization is broadened. In the frozen state, lipid droplets inside cumulus-oocyte complexes have a higher content of triglyceride polymorphic β and β′ phases than estimated for mature oocytes and early embryos. For the first time, to our knowledge, the temperature evolution of the phase state of lipid droplets is examined. Raman spectroscopy is proved to be a promising tool for in situ monitoring of the lipid phase state in a single embryo/oocyte during its freezing.

Investigation on the Nanomechanics of Liposome Adsorption on Titanium Alloys: Temperature and Loading Effects.

The mechanical properties of liposomes, determined by the lipid phase state at ambient temperature, have a close relationship with their physiological activities. Here, atomic force microscopy (AFM) was used to produce images and perform force measurements on titanium alloys at two adsorbed temperatures. The mechanical properties were evaluated under repeated loading and unloading, suggesting a better reversibility and resistance of gel phase liposomes. The liquid phase liposomes were irreversibly damaged during the first approach while the gel phase liposomes could bear more iterations, resulting from water flow reversibly going across the membranes. The statistical data offered strong evidence that the lipid membranes in the gel phase are robust enough to resist the tip penetration, mainly due to their orderly organization and strong hydrophobic interactions between lipid molecules. This work regarding the mechanical properties of liposomes with different phases provides guidance for future clinical applications, such as artificial joints.

Presence and persistence of a highly ordered lipid phase state in the avian stratum corneum.

To survive high temperatures in a terrestrial environment, animals must effectively balance evaporative heat loss and water conservation. In passerine birds, cutaneous water loss (CWL) is the primary avenue of water loss at thermoneutral temperatures and increases slightly as ambient temperature increases, indicating a change in the permeability of the skin. In the stratum corneum (SC), the outermost layer of the skin, lipids arranged in layers called lamellae serve as the primary barrier to CWL in birds. The permeability of these lamellae depends in large part on the ability of lipid molecules to pack closely together in an ordered orthorhombic phase state. However, as temperature increases, lipids of the SC become more disordered, and may pack in more permeable hexagonal or liquid crystalline phase states. In this study, we used Fourier transform infrared spectroscopy to monitor the phase state of lipids in the SC of house sparrows (Passer domesticus) at skin temperatures ranging from 25 to 50°C. As temperature increased, lipids became slightly more disordered, but remained predominantly in the orthorhombic phase, consistent with the small increase in CWL observed in house sparrows as ambient temperature increases. These results differ considerably from studies on mammalian SC, which find a predominantly hexagonal arrangement of lipids at temperatures above 37°C, and the increased order in avian SC may be explained by longer lipid chain length, scarcity of cholesterol and the presence of cerebrosides. Our results lend further insight into the arrangement and packing of individual lipid molecules in avian SC.

Raman spectroscopy reveals the lipid phase transition in preimplantation mouse embryos during freezing

Although lipid phase transition is believed to be among the major damaging factors in oocytes and preimplantation embryos cryopreservation, lack of the appropriate experimental methods limits investigation of this phenomenon. Herein, we demonstrate the capabilities of Raman spectroscopy to detect the lipid phase transition within the freezing preimplantation mouse embryos. We exploit the sensibility of antisymmetric CH2 Raman peak to the phase state of lipids. It is shown that during the freezing of the mouse embryos the lipid phase transition occurs at the temperatures between −7 and 0 °C. Similar temperature dependences of CH2 mode intensities are found for lipids in the preimplantation embryos and a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, implying the similarity in the occupation rules of conformational states. Raman spectroscopy is considered as a method of choice to study the lipid phase transition during preimplantation mammalian embryos freezing and cryopreservation.

Effect of Lipid Surface Composition on the Formation and Structure of Fibrin Clots.

We studied the influence of lipid surface composition on the kinetics of fibrin clot formation and its structure. It was shown that lipid surface affects all phases of fibrin polymerization and chances clot morphology. The magnitude and character of the effect depend on the charge and phase state of lipids that determine the interaction of fibrinogen with the lipid surface and its conformational changes, which modulated the process of fibrinogen conversion into fibrin and, as a result, the formation and morphology of the fibrin clot.

Structural templating of J-aggregates: Visualizing bis(monoacylglycero)phosphate domains in live cells

Identifying the key structural and dynamical determinants that drive the association of biomolecules, whether in solution, or perhaps more importantly in a membrane environment, has critical implications for our understanding of cellular dynamics, processes, and signaling. With recent advances in high-resolution imaging techniques, from the development of new molecular labels to technical advances in imaging methodologies and platforms, researchers are now reaping the benefits of being able to directly characterize and quantify local dynamics, structures, and conformations in live cells and tissues. These capabilities are providing unique insights into association stoichiometries, interactions, and structures on sub-micron length scales. We previously examined the role of lipid headgroup chemistry and phase state in guiding the formation of pseudoisocyanine (PIC) dye J-aggregates on supported planar bilayers [Langmuir, 25, 10719]. We describe here how these same J-aggregates can report on the in situ formation of organellar membrane domains in live cells. Live cell hyperspectral confocal microscopy using GFP-conjugated GTPase markers of early (Rab5) and late (Rab7) endosomes revealed that the PIC J-aggregates were confined to domains on either the limiting membrane or intralumenal vesicles (ILV) of late endosomes, known to be enriched in the anionic lipid bis(monoacylglycero)phosphate (BMP). Correlated confocal fluorescence - atomic force microscopy performed on endosomal membrane-mimetic supported planar lipid bilayers confirmed BMP-specific templating of the PIC J-aggregates. These data provide strong evidence for the formation of BMP-rich lipid domains during multivesicular body formation and portend the application of structured dye aggregates as markers of cellular membrane domain structure, size, and formation.

Effects of hydroxy-xanthones on dipalmitoylphosphatidylcholine lipid bilayers: A theoretical and experimental study

Xanthones and derivatives are natural active compounds whose interest has been increased due to its several pharmacological effects. In this work, effects of hydroxy-xanthones on the physicochemical properties of dipalmitoylphosphatidylcholine (DPPC) liposomes have been investigated in terms of lipid bilayer fluidity, by means of molecular dynamics simulations and temperature dependence of zeta potential studies. Experimental results predict, in good agreement with simulations, that xanthones are able to be incorporated into DPPC liposomes with certain localization, fluidizing the bilayer. Both effects, localization and fluidity were found to be dependent of the number of hydroxilic substituents of the xanthone and the lipid phase state.