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Dehydrodieugenol, a neolignan isolated from the Brazilian plant Nectandra leucantha (Lauraceae) with reported antiprotozoal and anticancer activity, was incorporated in Langmuir monolayers of selected lipids as cell membrane models, aiming to comprehend its action mechanism at the molecular level. The interaction of this compound with the lipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylserine (DPPS), and dipalmitoylphosphatidylglycerol (DPPG) was inferred through tensiometry, infrared spectroscopy, and Brewster angle microscopy. The interactions had different effects depending on the chemical nature of the lipid polar head, with expansion for DPPC monolayers, condensation for DPPE, and expansion (at low surface pressures) followed by the overlap of the isotherms (at high surface pressure values) for DPPS and DPPG. Effects caused by dehydrodieugenol in the negatively charged lipids were distinctive, which was also reflected in the hysteresis assays, surface potential-area isotherms, and rheological measurements. Infrared spectroscopy indicated that the drug interaction with the monolayer affects not only the polar groups, but also the acyl lipid chains for all lipids. These results pointed to the fact that the interaction of the drug with lipid monolayers at the air-water interface is modulated by the lipid composition, mainly considering the polar head of the lipids, as well as the hydrophobicity of the lipids and the drug. As negatively charged lipids pointed to distinctive interaction, we believe this can be related to the antiprotozoal and anticancer properties of the compound.

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Dengue virus infection depends on its fusion with the host membrane, where the binding occurs through interaction between proteins on the virus cell surface and specific viral receptors on target membranes. This process is mediated by the fusion peptide located between residues 98 and 112 (DRGWGNGCGLFGKGG) that forms a loop in domain II of dengue E glycoprotein. In this study, we evaluated the role of fusion peptide surrounding regions (88-97 and 113-123) of the Dengue 2 subtype on its interaction with the membrane and fusion activity. These sequences are important to stabilize the fusion peptide loop and increase fusion activity. Three peptides, besides the fusion peptide, were synthesized by SPPS using the Fmoc chemical approach. The first contains the fusion peptide and the C-terminal region of the loop (sequence 98-123); another contains the N-terminal region (88-112) and the larger peptide contains both regions (88-123). The peptides were able to interact with a model membrane. Differences in morphology of the monolayer promoted by the peptides were assessed by Brewster Angle Microscopy (BAM). Our data indicated that the C-terminal region of fusion peptide loop is more efficient in promoting fusion and interacting with the membrane than the N-terminal sequence, which is responsible for the electrostatic initial interaction. We propose a 2-step mechanism for the interaction of the dengue virus fusion peptide with the host membrane, where the N-terminal sequence docks electrostatically on the headgroups and then the C-terminal interacts via hydrophobic forces in the acyl chains.

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HYPOTHESIS: The interaction of chitosan, a natural biopolymer with various biomedical applications, with lipid Langmuir films has been widely investigated as a simple model for cell membranes. However, to ensure polymer solubility, up to now only acidic subphases with pH significantly below biological fluids have been used. To increase the biological significance of these investigations, here we evaluated the effects of two chitosan derivatives (low molecular weight - CH, and positively charged - CH-P40) on phospholipid films (either zwitterionic DPPC or anionic DPPG) using phosphate buffered saline solutions (PBS) as a subphase. EXPERIMENTS: Surface pressure - area (π-A) isotherms were used to evaluate the expansion and changes in film elasticity, while Sum-Frequency Generation (SFG) vibrational spectroscopy provided information about the chain conformation of lipids. FINDINGS: It was found that chitosans caused a small expansion of the DPPC film by its insertion within the monolayer. In contrast, they distinctly expanded DPPG monolayers by both chitosan insertion within the lipid monolayer and by interacting with the anionic head group. Therefore, PBS buffer can be used as a subphase for more biologically relevant studies of chitosan interactions with Langmuir films, shedding light on why chitosan is antibacterial but not toxic to mammals, as the interaction mechanism depends on lipid headgroup charge.

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ß-Galactosylsphingosine or psychosine (PSY) is a single chain sphingolipid with a cationic group, which is degraded in the lysosome lumen by ß-galactosylceramidase during sphingolipid biosynthesis. A deficiency of this enzyme activity results in Krabbe's disease and PSY accumulation. This favors its escape to extralysosomal spaces, with its pH changing from acidic to neutral. We studied the interaction of PSY with model lipid membranes in neutral conditions, using phospholipid vesicles and monolayers as classical model systems, as well as a complex lipid mixture that mimics the lipid composition of myelin. At pH 7.4, when PSY is mainly neutral, it showed high surface activity, self-organizing into large structures, probably lamellar in nature, with a CMC of 38 ±â€¯3 µM. When integrated into phospholipid membranes, PSY showed preferential partition into disordered phases, shifting phase equilibrium. The presence of PSY reduces the compactness of the membrane, making it more easily compressible. It also induces lipid domain disruption in vesicles composed of the main myelin lipids. The surface electrostatics of lipid membranes was altered by PSY in a complex manner. A shift to positive zeta potential values evidenced its presence in the vesicles. Furthermore, the increase of surface potential and surface water structuring observed may be a consequence of its location at the interface of the positively charged layer. As Krabbe's disease is a demyelinating process, PSY alteration of the membrane phase state, lateral lipid distribution and surface electrostatics appears important to the understanding of myelin destabilization at the supramolecular level.

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A rather widespread idea on the functional importance of sphingolipids in cell membranes refers to the occurrence of ordered domains enriched in sphingomyelin and ceramide that are largely assumed to exist irrespective of the type of N-acyl chain in the sphingolipid. Ceramides and sphingomyelins are the simplest kind of two-chained sphingolipids and show a variety of species, depending on the fatty acyl chain length, hydroxylation, and unsaturation. Abundant evidences have shown that variations of the N-acyl chain length in ceramides and sphingomyelins markedly affect their phase state, interfacial elasticity, surface topography, electrostatics, and miscibility, and that even the usually conceived "condensed" sphingolipids and many of their mixtures may exhibit liquid-like expanded states. Their lateral miscibility properties are subtlety regulated by those chemical differences. Even between ceramides with different acyl chain length, their partial miscibility is responsible for a rich two-dimensional structural variety that impacts on the membrane properties at the mesoscale level. In this review, we will discuss the miscibility properties of ceramide, sphingomyelin, and glycosphingolipids that differ in their N-acyl or oligosaccharide chains. This work is a second part that accompanies a previous overview of the properties of membranes formed by pure ceramides or sphingomyelins, which is also included in this Special Issue.

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Polybia-MP1 or simply MP1 (IDWKKLLDAAKQIL-NH2) is a peptide with broad-spectrum bactericidal activity and a strong inhibitory effect against cancer cells. The aim of this work was to evaluate the effect of biophysical properties such as membrane texture and film thickness on MP1 interaction with neutral and anionic lipid membranes. For this purpose, we first explored the peptide's surface behavior. MP1 showed high surface activity, adsorbing onto bare air/aqueous interfaces up to higher surface pressures than the collapse pressure of MP1 Langmuir films. The MP1-lipid membrane interaction was studied using Langmuir phosphatidylcholine and phosphatidylserine (PS) monolayers as model membrane systems. PS was chosen since this negatively charged lipid was found predominantly on the outer leaflet of tumor cells, and it enhances MP1 activity for PS-containing membranes to a greater extent than for other negatively charged lipids. MP1 incorporated into anionic PS monolayers, which show a liquid-expanded (LE) phase or LE-liquid-condensed (LC) phase coexistence, up to lipid-packing densities higher than those of cell membranes. The mixed lipid/MP1 films were explored by Brewster angle microscopy and atomic force microscopy. MP1 partitioned preferentially into the LE phase state of PS films, and were thus excluded from the coexisting LC phase. This interaction had strong electrostatic bases: in pure water, the lipid-peptide interaction was strong enough to induce formation of reversible lipid-peptide 3D structures associated with the interface. MP1 incorporation into the LE phase was accompanied by a shift of the phase transition pressure to higher values and a thinning of the lipid film. These results showed a clear correlation between peptide penetration capacity and the presence or induction of the thin LE phase. This capacity to regulate membrane physical properties may be of relevance in the binding, incorporation and membrane selectivity of this promising antitumor peptide.

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Ceramides, the simplest kind of two-chained sphingolipids, contain a single hydroxyl group in position 1 of the sphingoid base. Sphingomyelins further contain a phosphocholine group at the OH of position 1 of ceramide. Ceramides and sphingomyelins show a variety of species depending on the fatty acyl chain length, hydroxylation, and unsaturation. Because of the relatively high transition temperature of sphingomyelin compared to lecithin and, particularly, of ceramides with 16:0-18:0 saturated chains, a widespread idea on their functional importance refers to formation of rather solid domains enriched in sphingomyelin and ceramide. Frequently, and especially in the cell biology field, these are generally (and erroneously) assumed to occur irrespective on the type of N-acyl chain in these lipids. This is because most studies indicating such condensed ordered domains employed sphingolipids with acyl chains with 16 carbons while scarce attention has been focused on the influence of the N-acyl chain on their surface properties. However, abundant evidence has shown that variations of the N-acyl chain length in ceramides and sphingomyelins markedly affect their phase state, interfacial elasticity, surface topography, electrostatics and miscibility and that, even the usually conceived "condensed" sphingolipids and many of their mixtures, may exhibit liquid-like expanded states. This review is a summarized overview of our work and of related others on some facts regarding membranes composed of single molecular species of ceramide and sphingomyelin. A second part is dedicated to discuss the miscibility properties between species of sphingolipids that differ in N-acyl and oligosaccharide chains.

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Langmuir monolayers at the air/water interface are widely used as biomembrane models and for amphiphilic molecules studies in general. Under controlled intermolecular organization (lateral molecular area), surface pressure, surface potential, reflectivity (R) and other magnitudes can be precisely determined on these planar monomolecular films. However, some physical parameters such as the refractive index of the monolayer (n) still remain elusive. The refractive index is very relevant because (in combination with R) it allows for the determination of the thickness of the film. The uncertainties of n determine important errors that propagate non-linearly into the calculation of monolayers thickness. Here we present an analytical method for the determination of n in monolayers based on refractive index matching. By using a Brewster angle microscopy (BAM) setup and monolayers spread over subphases with variable refractive index (n2), a minimum in R is search as a function of n2. In these conditions, n equals n2. The results shown correspond to monolayers of myelin lipids. The n values remain constant at 1.46 upon compression and equals the obtained value for myelin lipid bilayers in suspension. The values for n and R allow for the determination of thickness. We establish comparisons between these thicknesses for the monolayer and those obtained from two X-ray scattering techniques: 1) GIXOS for monolayers at the air/water interface and 2) SAXS for bilayers in bulk suspension. This allows us to conclude that the thickness that we measure by BAM includes the apolar and polar headgroup regions of the monolayer.

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The cyclic ketones, thujone and dihydrocarvone, are lipophilic components of essential oils extracted from different plants, which have proven insecticidal activity. The GABAA receptor is activated by the neurotransmitter GABA and is the action site of widely used neurotoxic pesticides. Many compounds that regulate GABAA receptor function interact with membrane lipids, causing changes in their physical properties and consequently, in the membrane dynamic characteristics that modulate receptor macromolecules. In the present study, the biophysical effects of thujone (a gabaergic reference compound) and dihydrocarvone (structurally very similar) were explored by using monomolecular films of DPPC as a model membrane system, to gain insight into membrane-drug interaction. The compression isotherms showed that both ketones expand the DPPC isotherms and increase membrane elasticity. They penetrate the monolayer but their permanence depends on the possibility of establishing molecular interactions with the film component, favored by defects present in the membrane at the phase transition. Finally, by using Brewster angle microscopy (BAM) as a complementary technique for direct visualization of the study films, we found that incorporating ketone seems to reduce molecular repulsion among phospholipid headgroups. Our results reinforce the notion that changes in membrane mechanics may be occurring in the presence of the assayed ketones, suggesting that their interaction with the receptor's surrounding membrane may modulate or affect its functionality, possibly as part of the mechanism of the bioactivity described for thujone and DHC.

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Unique species of ceramide (Cer) with very-long-chain polyunsaturated fatty acid (VLCPUFA), mainly 28-32 carbon atoms, 4-5 double bonds, in nonhydroxy and 2-hydroxy forms (n-V Cer and h-V Cer, respectively), are generated in rat spermatozoa from the corresponding sphingomyelins during the acrosomal reaction. The aim of this study was to determine the properties of these sperm-distinctive ceramides in Langmuir monolayers. Individual Cer species were isolated by HPLC and subjected to analysis of surface pressure, surface potential, and Brewster angle microscopy (BAM) as a function of molecular packing. In comparison with known species of Cer, n-V Cer and h-V Cer species showed much larger mean molecular areas and increased molecular dipole moments in liquid expanded phases, which suggest bending and partial hydration of the double bonded portion of the VLCPUFA. The presence of the 2-hydoxyl group induced a closer molecular packing in h-V Cer than in their chain-matched n-V Cer. In addition, all these Cer species showed liquid-expanded to liquid-condensed transitions at room temperature. Existence of domain segregation was confirmed by BAM. Additionally, thermodynamic analysis suggests a phase transition close to the physiological temperature for VLCPUFA-Cers if organized as bulk dispersions.

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Ascorbyl palmitate (ASC16) is an anionic amphiphilic molecule of pharmacological interest due to its antioxidant properties. We found that ASC16 strongly interacted with model membranes. ASC16 penetrated phospholipid monolayers, with a cutoff near the theoretical surface pressure limit. The presence of a lipid film at the interface favored ASC16 insertion compared with a bare air/water surface. The adsorption and penetration time curves showed a biphasic behavior: the first rapid peak evidenced a fast adsorption of charged ASC16 molecules to the interface that promoted a lowering of surface pH, thus partially neutralizing and compacting the film. The second rise represented an approach to the equilibrium between the ASC16 molecules in the subphase and the surface monolayer, whose kinetics depended on the ionization state of the film. Based on the Langmuir dimiristoylphosphatidylcholine+ASC16 monolayer data, we estimated an ASC16 partition coefficient to dimiristoylphosphatidylcholine monolayers of 1.5×10(5) and a ΔGp=-6.7kcal·mol(-1). The rheological properties of the host membrane were determinant for ASC16 penetration kinetics: a fluid membrane, as provided by cholesterol, disrupted the liquid-condensed ASC16-enriched domains and favored ASC16 penetration. Subphase pH conditions affected ASC16 aggregation in bulk: the smaller structures at acidic pHs showed a faster equilibrium with the surface film than large lamellar ones. Our results revealed that the ASC16 interaction with model membranes has a highly complex regulation. The polymorphism in the ASC16 bulk aggregation added complexity to the equilibrium between the surface and subphase form of ASC16, whose understanding may shed light on the pharmacological function of this drug.

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