Miscibility of binary monolayers at the air-water interface and interaction of protein with immobilized monolayers by surface plasmon resonance technique

The miscibility and stability of the binary monolayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and cationic dioctadecyldimethylammonium bromide (DOMA) at the air-water interface and the interaction of ferritin with the immobilized monolayers have been studied in detail using surface pressure-area isotherms and surface plasmon resonance technique, respectively. The surface pressure-area isotherms indicated that the binary monolayers of DPPC and DOMA at the air-water interface were miscible and more stable than the monolayers of the two individual components. The surface plasmon resonance studies indicated that ferritin binding to the immobilized monolayers was primarily driven by the electrostatic interaction and that the amount of adsorbed protein at saturation was closely related not only to the number of positive charges in the monolayers but also to the pattern of positive charges at a given mole fraction of DOMA. The protein adsorption kinetics was determined by the properties of the monolayers (i.e., the protein-monolayer interaction) and the structure of preadsorbed protein molecules (i.e., the protein-protein interaction).     

Interactions of Pluronics with phospholipid monolayers at the air-water interface

Pluronics are triblock copolymers which are extensively applied excipients shown to interact with cell membranes. The aim of our study was to apply monolayer techniques and epifluorescence microscopy to investigate the interaction behavior between selected Pluronics and phospholipid monolayers which serve as a model of cell membranes. The results showed that Pluronic L61 with hydrophobic proportions much larger than those of F68 demonstrated condensed film-like surface behavior while F68 exhibited more expanded behavior. The increments of surface pressure and the changes of image were more obvious in adding Pluronic L61 than F68 to the subphase of dipalmitoylphosphatidylcholine (DPPC) monolayers, which indicated that the interaction may be related to van der Waals forces and hydrophobic interaction. Pluronics selected with higher hydrophobicities demonstrated larger surface activities and penetration abilities while being added to the subphase of DPPC and dimyristoylphosphatidylcholine (DMPC) monolayers. Pluronic P85 and F68 were found to be squeezed to subphase at higher surface pressures, which may be attributed to their relatively higher hydrophilicities.     

The determination of work of compression of protein monolayers at the air-water interface

Summary In the present study, monolayers of various proteins were investigated at the air-water interface. The work of compression,W _c (Helmholtz free energy) has been determined from the surface pressure-area compression isotherms. A linear relationship was found betweenW _c and the amount of protein present at the surface. Further, it is shown that this relation holds both for completely unfolded (Bovine-serum-albumin, Ovalbumin) and for incompletely unfolded (Transferrin, Myoglobin) proteins.β-Lipoprotein isotherms also gave a similar dependence. It is further shown that the amount of protein lost into the subphase can be determined from a plot ofW _c versus protein added at the interface. The results are discussed in relation to the constitution of protein molecules at the surface.     

Interactions of anthracyclines with zwitterionic phospholipid monolayers at the air-water interface

The present note describes the use of surface pressure measurements (Langmuir monolayer technique) for the analysis of interactions of two different anthracyclines (adriamycin and daunorubicin) with a non-ionic, zwitterionic phospholipid monolayer, at the air-water interface. Because the surface membrane of the cell is the first barrier encountered by the anthracyclines in the treatment of cancer, drug-membrane interactions studied in model (monolayers or bilayers) and natural systems play an important role in the understanding of the bioactivity properties of these molecules. We report here the rate constants of the adsorption process of adriamycin and daunorubicin in the presence of a zwitterionic phospholipid monolayer at the air-water interface. Because interactions with the lipid monolayer strongly depend on the molecular packing of the lipid, we investigated this process at a relatively low surface pressure (7 mN/m), the interactions being favoured by the gaseous and liquid expanded structure of the lipid monolayer. The apparent molecular area of these molecules during the insertion into the lipid film and their interactions with the phospholipid polar head groups was evaluated and the estimated percentage of anthracyclines at the interface after adsorption into the lipid monolayer is briefly discussed. The rate constants for the adsorption and desorption process at the water-monolayer interface have been calculated on the basis of a single-exponential model. The observed difference of these parameters for daunorubicin and adriamycin suggests a different interaction of these anthracyclines during the adsorption to and/or penetration across the phospholipid monolayer.     

Mixed monolayers of poly-alanine and lipids at the air-water interface

Summary The mixed monolayers of poly-alanine + stearyl alcohol and poly-alanine + cholesterol were studied at the air-water interface. In the mixed monolayers the surface pressure-area isotherms showed three collapse states. The first and the third collapse pressures were identical in magnitude with the collapse pressures of pure components. The intermediate collapse pressure in the poly-alanine + stearyl alcohol was found to be ca. 5 dyne/cm higher than that was observed in the poly-alanine + cholesterol system. Further, the mixed films in both systems were found to show no deviation from the ideality rule. The magnitude o f the intermediate collapse state is shown to be related to the van der Waals forces present in the lipid films.With 6 figures     

Close-packed monolayers of charged Janus-type nanoparticles at the air-water interface

Two DNA-block copolymers, poly(caprolactone)-DNA and poly(methyl metacrylate)-DNA, were synthesized by conjugation of a short single strand of DNA (12 or 22 mer) to a single reactive group at one end of the synthetic polymer. These polymers self-assemble in water, without the need of any cosolvent, forming micelle-like aggregates that were imaged by TEM. The solution behavior of the bioconjugated polymers was investigated by surface tension measurements. In the direction of dilution, the surface tension was measured using a down-scaled Wilhelmy plate method. To proceed in the reverse direction (concentration), we measured the surface tension of a sessile drop during its evaporation. This latter method was firstly validated using ionic and non-ionic surfactants, including polymeric surfactants. It was then applied to investigate the unimer to micelles transition of the DNA-block copolymers. In all cases, a reversible transition was observed demonstrating the existence of a critical micellar concentration, close to 0.01 mmol L⁻¹ for all the conjugates. The CMC was only slightly influenced by the length of the hydrophilic DNA block.     

Structural analysis of PEO-PBO copolymer monolayers at the air-water interface

X-ray reflectivity (XR) and grazing incidence X-ray diffraction (GIXD) have been used to examine an oxyethylene-b-oxybutylene (E(23)B(8)) copolymer film at the air-water interface. The XR data were fitted using both a one- and a two-layer model that outputted the film thickness, roughness, and electron density. The best fit to the experimental data was obtained using a two-layer model (representing the oxyethylene and oxybutylene blocks, respectively), which showed a rapid thickening of the copolymer film at pressures above 7 mN/m. The large roughness values found indicate a significant degree of intermixing between the blocks and back up the GIXD data, which showed no long range lateral ordering within the layer. It was found from the electron density model results that there is a large film densification at 7 mN/m, possibly suggesting conformational changes within the film, even though no such change occurs on the pressure-area isotherm at the same surface pressure.     

Adsorption of DNA to octadecylamine monolayers at the air-water interface

In this work, surface properties of octadecylamine (ODA) monolayers in the presence of different concentrations of calf thymus DNA in the aqueous subphase covering a range of 2-8μM have been investigated. The increase of DNA concentration is accompanied by a marked increment in the expansion of the corresponding isotherms. In addition, there is a change in the profile of the isotherms ranging from an abrupt liquid-solid transition for the lipid monolayer on pure water to a slow condensation of the monolayer in a liquid state when DNA is added to the subphase, demonstrating the effective adsorption of the polynucleotide to the long chain amine monolayer. Additional phase transitions appear in the isotherms upon addition of sufficient amount of DNA, revealing the existence of specific processes such as folding or squeezing out of the DNA. This system is, however, highly reversible during compression-expansion cycles due to the strong interaction between the two components. These results are also supported by Brewster Angle Microscopy (BAM) images showing significant changes in the morphology of the film. Integral reflectivity of the BAM microscope has been used to study both isotherms themselves and the kinetic process of DNA inclusion into the lipid-like ODA monolayer. This parameter has been proven to be very effective for quantification of the monolayer processes showing high consistency with the compressibility and kinetics results.     

Protein-directed assembly of binary monolayers at the interface and surface patterns of protein on the monolayers

Ferritin-directed assembly of binary monolayers of zwitterionic dipalmitoylphosphatidylcholine and cationic dioctadecyldimethylammonium bromide (DOMA) at the interface and surface patterns of ferritin on the monolayers have been investigated using a combination of infrared reflection absorption spectroscopy, surface plasmon resonance, and atomic force microscopy. Ferritin binding to the binary monolayers at the air-water interface at the surface pressure 30 mN/m, primarily driven by the electrostatic interaction, gives rise to a change in tilt angle of hydrocarbon chains from 15 degrees +/- 1 degrees to 10 degrees +/- 1 degrees with respect to the normal of the monolayer at the mole fraction of DOMA (XDOMA) of 0.1. The chains at XDOMA = 0.3 are oriented vertical to the water surface before and after protein binding. A new mechanism for protein binding to the binary monolayers is proposed. The secondary structures of the adsorbed ferritin are prevented from changing to some extent due to the existence of the monolayers. The amounts of the bound protein on the monolayers at the air-water interface are increased in comparison with those on the pre-immobilized monolayers at low XDOMA. The increased amounts and different patterns of the adsorbed protein at the monolayers are mostly attributed to the formation of multiple binding sites available for ferritin, which is due to the lateral reorganization of the lipid components in the monolayers induced by the protein in the subphase. The created multiple binding sites on the monolayer surfaces through the protein-directed assembly can be preserved for subsequent protein binding.     

Crystalline self-assembly into monolayers of folded oligomers at the air-water interface

Insertion of the 1,3-bis(ethynylene)benzene unit as a rigid spacer into a linear alkyl chain, thus separating the two resulting stems by 9 A. induces chain folding at the air-water interface. These folded molecules self-assemble into crystalline monolayers at this interface, with the plane of the folding unit almost perpendicular to the water surface, as determined by synchrotron grazing-incidence X-ray diffraction. Three distinct molecular shapes, of the types U, inverted U, and M, were obtained in the two-dimensional crystalline state, depending upon the number of spacer units, and the number and position of the hydrophilic groups in the molecule. The molecules form ribbons with a higher crystal coherence in the direction of stacking between the molecular ribbons, and a lower coherence along the ribbon direction. A similar molecule, but with a spacer unit that imposes a 5 A separation between alkyl chains, yields the conventional herringbone arrangement.     

Metal ion coordination with an asymmetric fan-shaped dendrimer at the air-water interface

Metal coordination to monolayers of 4-{10-[4-(3,5-bis-benzyloxy)-phenyl]-anthracen-9-yl}-benzoic acid ([G1-An]-CO(2)H, G1) and 4-(10-{4-[3,5-bis-(3,5-bis-benzyloxy)-benzyloxy]-phenyl}-anthracen-9-yl)-benzoic acid ([G2-An]-CO(2)H, G2) at the air-water interface and to Langmuir-Blodgett (LB) films was investigated using surface pressure-area isotherms, ultraviolet-visible (UV-vis) spectroscopy, atomic force microscopy (AFM), and X-ray reflectivity (XRR). Surface pressure-area isotherms show that G1 and G2 have different limiting areas according to the type of subphase. The limiting area of G1 and G2 increased more with Al(3+) than with Eu(3+) in the subphase. This result indicates that the hydrophilic core group is anchored to ions in the water via bidentate chelates with the carboxylate oxygen atoms of G1 and G2. Circular domains and aggregates were observed for the LB film. The different behavior of Eu(3+) and Al(3+) complexes is originated from the intrinsic nature of the ion, i.e., coordination number.     

Interaction of the cationic peptide bactenecin with mixed phospholipid monolayers at the air-water interface

The initial mechanism by which antimicrobial peptides target microbes occurs via electrostatic interactions; however, the mechanism is not well understood. We investigate the interaction of the antimicrobial peptide bactenecin with a 50:50 w:w% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) phospholipid mixture at the air-water interface with different NaCl concentrations (0.01, 0.05, 0.1, 0.5 M) in the subphase. A larger shift of DPPC:DMPG isotherms was obtained for 0.1 M salt concentration at lower and higher pressures, demonstrating the influence of the negative charge of DMPG molecules and the screening of the electrostatic interaction by the salt concentration. Raman spectroscopy of monolayers demonstrated the presence of cysteine-cysteine bridges in bactenecin loops. The peptide adsorption in DPPC:DMPG monolayers observed by AFM images suggests a self-assembled aggregation process, starting with filament-like networks. Domains similar to carpets were formed and pore structures were obtained after a critical peptide concentration, according to the carpet model.     

Incorporation and exclusion of long chain alkyl halides in fatty acid monolayers at the air-water interface

Mixed monolayers of deuterated palmitic acid C(15)D(31)COOH (dPA) and deuterated stearic acid C(17)D(35)COOH (dSA) with 1-bromoalkanes of different alkyl chain length (C(4) to C(16)) at the air-water interface were investigated. Alkanes and 1-chlorohexadecane ClC(16)H(33) (ClHex) were also studied to compare the effects of the halogen on the mixed monolayers. Surface pressure-area isotherms and Brewster angle microscopy (BAM) were used to obtain the organization and phase behavior, providing a macroscopic view of the mixed monolayers. A molecular-level understanding of the interfacial molecular organization and intermolecular interactions was obtained by using vibrational sum frequency generation (SFG) spectroscopy and infrared reflection-absorption spectroscopy (IRRAS). It was found that from the alkyl halide molecules investigated 1-bromopentadecane, BrC(15)H(31) (BrPent), 1-bromohexadecane, BrC(16)H(33) (BrHex), and ClHex incorporate into the fatty acid monolayers. Alkanes of 15- and 16-carbon chain length do not incorporate into the fatty acid monolayer, which suggests that the halogen is needed for incorporation. Isotherms and spectra suggest that BrHex molecules are squeezed out, or excluded, from the fatty acid monolayer as the surface pressure is increased, while BAM images confirm this. Additionally, SFG spectra reveal that the alkyl chains of both fatty acids (dPA and dSA) retain an all-trans conformation after the incorporation of alkyl halide molecules. BAM images show that at low surface pressures BrHex does not affect the two-dimensional morphology of the dPA and dSA domains and that BrHex is miscible with dPA and dSA. We also present for the first time BAM images of BrHex deposited on a water surface, which reveal the formation of aggregates while the surface pressure remains unchanged from that of neat water.     

Interactions of L-arginine with Langmuir monolayers of di-n-dodecyl hydrogen phosphate at the air-water interface

The surface phase behavior of di-n-dodecyl hydrogen phosphate (DDP) in Langmuir monolayer and its interactions with L-arginine (L-arg) have been investigated by measuring pi-A isotherms with a film balance and observing monolayer morphology with a Brewster angle microscopy (BAM). The DDP monolayers on pure water show a first-order liquid expanded-liquid condensed (LE-LC) phase transition and form fingering LC domains having uniform brightness at different temperatures. At 15 degrees C, the pi-A isotherms on pure water and on different concentration solutions of L-arg show a limiting molecular area at approximately 0.50 nm(2)/molecule. With increasing the subphase concentration of L-arg up to 4.0 x 10(-4)M, the LE and the LE-LC coexistence regions shift to larger molecular areas and higher surface pressures, respectively. With a further increase in the concentration of L-arg beyond this critical concentration, these isotherms show little or no more expansion. These results have been explained by considering the fact that the L-arg undergoes complexation with the DDP to form L-arg-DDP that remains in equilibrium with the components at the air-water interface. As the concentration of L-arg in the subphase increases, the equilibrium shifts towards the complex. At a concentration of L-arg > or =4.0 x 10(-4)M, the DDP monolayers get saturated and show the characteristics of the new amphiphile, L-arg-DDP. BAM is applied to confirm the above results. When the concentration of the L-arg is <4.0 x 10(-4)M, domains always start forming at an area of approximately 0.64 nm(2)/molecule, which is the critical molecular area for the phase transition in the DDP monolayers on pure water. In contrast, when the monolayers are formed on a solution containing > or =4.0 x 10(-4)M L-arg, comparatively smaller size domains are formed after the appearance of a new cusp point at approximately 0.55 nm(2)/molecule. With an increase in the concentration of L-arg in the subphase, the size of the domains decreases indicating that the fraction of the DDP gradually decreases, whereas the fraction of the complex gradually increases. In addition, a very simple procedure for determination of the stability constant, which is 2.6 x 10(4)M(-1) at 15 degrees C, has been suggested.     

In situ characterization of functional purple membrane monolayers at the air-water interface

The purple membrane (PM) of Halobacterium salinarum contains a single type of protein, bacterio-rhodopsin (bR), which is a member of the seven alpha-helices transmembrane protein family. This protein is a photoactive proton pump, translocating one proton from the cytoplasmic to the extracellular side of the PM per photon absorbed. bR is found in trimers in PM, where they are assembled in a two-dimensional hexagonal lattice. We show herein that stable and functional films can be built in monolayers at the air-water interface by spreading aqueous suspensions of purified and native PM patches. In situ spectroscopic measurements at the air-water interface indicate that bR remains photoactive in this environment. Physical parameters of these PM films, such as protein molecular area, irreversible in-plane aggregation, z-axis orientation, film thickness, and surface roughness, were determined from surface pressure and surface potential-area isotherms, fluorescence spectroscopy, and X-ray reflectivity at the air-water interface. We find that PM do form organized monolayers of membranes, with an optimal packing density at a surface pressure of approximately 20 mN/m, although no preferential vectorial alignment, with respect to the plane normal to the membrane, can be detected from fluorescence quenching experiments.     

Phase behavior of 2,3-disubstituted methyl octadecanoate monolayers at the air-water interface

The phase behavior of 2,3-disubstituted methyl octadecanoate monolayers at the air-water interface is studied by film balance and a Brewster angle microscope (BAM). The comparison of the surface pressure-molecular area (pi-A) isotherms with the corresponding BAM images provides information on the phase behavior of the monolayers. Variations in the phase behavior of different 2,3-disubstituted methyl octadecanoate monolayers can be correlated with the size of the headgroups, the interactions between the polar molecular moieties and the subphase, and the intermolecular interactions. The enlarging of the headgroups makes forming a condensed monolayer difficult for the molecules, even after introduction of substituents giving rise to the formation of hydrogen bonds between the molecules, which may balance the steric repulsion and stabilize the monolayers. Model calculations of the two-dimensional lattice structure of the 2,3-disubstituted methyl octadecanoates on basis of the pg and p1 space group are performed and correspond well with the experimental results.     

Real-time imaging of crystallization in polylactide enantiomeric monolayers at the air-water interface

A newly developed planar array infrared reflection-absorption spectrograph (PA-IRRAS) offers significant advantages over conventional approaches including fast acquisition speed, excellent compensation for water vapor, and an excellent capacity for large infrared accessories, e.g., a water trough. In this study, the origin of stereocomplexation in a polylactide enantiomeric monolayer at the air-water interface was investigated using PA-IRRAS. PA-IRRAS was used as a probe to follow the real-time conformational changes associated with intermolecular interactions of polymer chains during the compression of the monolayers. It was found that a mixture of poly(D-lactic acid) (PDLA) and poly(L-lactic acid) (PLLA) (D/L) formed a stereocomplex when the two-dimensional monolayer developed at the air-water interface before film compression, indicating that there is no direct correlation between film compression and stereocomplexation. PA-IRRAS spectra of the stereocomplex exhibited distinct band shifts in crystalline sensitive components, e.g., the vas(C-O-C, h) mode, as well as amorphous-dependent components, e.g., the vs(C-O-C) mode, when compared with the spectra of PLLA alone. On the other hand, time-resolved PA-IRRAS spectra, which were obtained as the films were being compressed, revealed that both monolayers of PLLA and mixed PLLA/PDLA stereocomplex were crystallized into a 10(3)-helix and a 3(1)-helix, respectively, with a distinct band shift in crystalline sensitive components only. Fourier self-deconvolution of the spectra demonstrated that the band shift in crystalline sensitive components is correlated with the intermolecular interaction of polymer chains.     

Anchor-lipid monolayers at the air-water interface; prearranging of model membrane systems

Model membrane systems are gaining more and more interest both for basic studies of membrane-related processes as well as for biotechnological applications. Several different model systems have been reported among which the tethered bilayer lipid membranes (tBLMs) form a very attractive and powerful architecture. In all the proposed architectures, a control of the lateral organization of the structures at a molecular level is of great importance for an optimized preparation. For tBLMs, a homogeneous and not too dense monolayer is required to allow for the functional incorporation of complex membrane proteins. We present here an alternative approach to the commonly used self-assembly preparation. Lipids are spread on the air-water interface of a Langmuir film balance and form a monomolecular film. This allows for a better control of the lateral pressure and distribution for subsequent transfer to solid substrates. In this paper, we describe the properties of the surface monolayer, in terms of surface pressure, structure of the lipid molecule, content of lipid mixtures, temperature, and relaxations features. It is shown that a complete mixing of anchor-lipids and free lipids can be achieved. Furthermore, an increase of the spacer lengths and a decrease of the temperature lead to more compact films. This approach is a first step toward the fully controlled assembly of a model membrane system.     

Two-dimensional facets in Langmuir monolayers of 1-O-hexadecyl-rac-glycerol at the air-water interface

We study the surface phase behavior in Langmuir monolayers of 1-O-hexadecyl-rac-glycerol (C16G) by film balance and Brewster angle microscopy over a wide range of temperatures. A cusp point followed by a pronounced plateau region in the pressure-area (pi-A) isotherm indicates a first-order phase transition between a lower density liquid expanded (LE) phase and a higher density liquid condensed (LC) phase at the air-water interface. A wide variety of condensed domains are found to form just after the appearance of the cusp point. The observed surface morphology was compared with that of ethylene glycol mono-n-hexadecyl ether (C16E1) that bears an ethylene oxide (EO) unit in the head-group. As usually observed, the domains of C16E1 are found to be circular at lower temperatures and fractal at higher temperatures. Contrary to this usual behavior, the domains of C16G are found to be strip-like structures at lower temperatures, which attain increasingly compact shape as the temperature increases and finally attain faceted structures at > or = 25 degrees C. It is concluded that a higher degree of dehydration around the head-group region of C16G appreciably reduces the hydration-induced repulsive interactions between the head-groups and imparts to the molecules an increase in hydrophobicity, thereby a closer molecular packing. As a result, the molecules form increasingly compact domains as the temperature increases. Since the head-group of C16E1 is much smaller than that of C16G, dehydration effect cannot appreciably increase its hydrophobic character. Rather, increases in subphase temperature result in a decrease in the line tension of the interface giving fractal structures at higher temperatures. In addition, the changes in enthalpy (deltaH) and entropy (deltaS) values were also calculated to understand the thermodynamic nature of condensation of the molecules in the LE-LC transition region.