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.     

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.     

Mechanical properties of interfacial films formed by lysozyme self-assembly at the air-water interface

We present the first characterization of the mechanical properties of lysozyme films formed by self-assembly at the air-water interface using the Cambridge interfacial tensiometer (CIT), an apparatus capable of subjecting protein films to a much higher level of extensional strain than traditional dilatational techniques. CIT analysis, which is insensitive to surface pressure, provides a direct measure of the extensional stress-strain behavior of an interfacial film without the need to assume a mechanical model (e.g., viscoelastic), and without requiring difficult-to-test assumptions regarding low-strain material linearity. This testing method has revealed that the bulk solution pH from which assembly of an interfacial lysozyme film occurs influences the mechanical properties of the film more significantly than is suggested by the observed differences in elastic moduli or surface pressure. We have also identified a previously undescribed pH dependency in the effect of solution ionic strength on the mechanical strength of the lysozyme films formed at the air-water interface. Increasing solution ionic strength was found to increase lysozyme film strength when assembly occurred at pH 7, but it caused a decrease in film strength at pH 11, close to the pI of lysozyme. This result is discussed in terms of the significant contribution made to protein film strength by both electrostatic interactions and the hydrophobic effect. Washout experiments to remove protein from the bulk phase have shown that a small percentage of the interfacially adsorbed lysozyme molecules are reversibly adsorbed. Finally, the washout tests have probed the role played by additional adsorption to the fresh interface formed by the application of a large strain to the lysozyme film and have suggested the movement of reversibly bound lysozyme molecules from a subinterfacial layer to the interface.     

Subtilisin-catalyzed religation of proteolyzed hen egg-white lysozyme: investigation of the role of disulfides

Background: The use of proteases to form, instead of break, peptide bonds has expanded the repertoire of techniques available for protein semisynthesis. Several groups have previously reported the use of proteases in aqueous-organic solvents to form single amide bonds within proteins, but low yields and lengthy reaction times make this an impractical approach to protein synthesis. We recently found that proteolyzed triose phosphate isomerase can be re-ligated rapidly and efficiently by subtilisin, in mixed aqueous-organic solvent systems.Results: We now report the use of subtilisin to resynthesize hen egg-white lysozyme from a mixture of its proteolyzed fragments in high yield and with rapid reaction times. This enzymatic religation can also be achieved after reduction of the four disulfide bonds present in lysozyme, with the same efficiency as that observed for the disulfide-containing proteolysis mixture.Conclusions: For egg-white lysozyme, the subtilisin religation reaction can be used to re-synthesize a proteolyzed protein even after reduction of disulfide bonds. The utility of this reaction in more generalized protein semisynthesis reactions is currently being explored.     

Hydroxyl radical at the air-water interface

Interaction of the hydroxyl radical with the liquid water surface was studied using classical molecular dynamics computer simulations. From a series of scattering trajectories, the thermal and mass accommodation coefficients of OH on liquid water at 300 K were determined to be 0.95 and 0.83, respectively. The calculated free energy profile for transfer of OH across the air-water interface at 300 K exhibits a minimum in the interfacial region, with the free energy of adsorbtion (DeltaGa) being about 1 kcal/mol more negative than the hydration free energy (DeltaGs). The propensity of the hydroxyl radical for the air-water interface manifests itself in partitioning of OH radicals between the bulk water and the surface. The enhancement of the surface concentration of OH relative to its concentration in the aqueous phase suggests that important OH chemistry may be occurring in the interfacial layer of water droplets, aqueous aerosol particles, and thin water films adsorbed on solid surfaces. This has profound consequences for modeling heterogeneous atmospheric chemical processes.     

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.     

Surface rheology of hydrophobically modified PEG polymers associating with a phospholipid monolayer at the air-water interface

Surface rheology of irreversibly bound hydrophobically modified poly(ethylene glycol) (PEG) polymers (HMPEG) on a dipalmitoylphosphatidylcholine (DPPC) monolayer is investigated to determine attributes that may contribute to immune recognition. Previously, three comb-graft polymers (HMPEG136-DP3, HMPEG273-DP2.5, and HMPEG273-DP5) adsorbed on liposomes were examined for their strength of adsorption and protection from complement binding. The data supported an optimal ratio between the hydrophilicity of the PEG polymer and the number of hydrophobic anchors. The HMPEG polymers have different polymer brush thicknesses (4.2-5.9 nm) and levels of cooperativity (2.5-5 hydrophobes). The results indicate that an increased viscous force (above 0.25 mN s/m) at the surface may enable the polymers to shield liposomes from protein interactions. Similar rheological behavior is shown for all polymer architectures at low polymer surface coverage (0.5 mg/m2, in the mushroom regime), whereas at high surface coverage (>0.5 mg/m2, in the brush regime), we observe a structural dependence of the surface viscous forces at 40 mN/m. This threshold correlates with a 92% decrease in complement protein binding for liposomes coated with 1 mg/m2 HMPEG273-DP5. This may suggest that surface viscous forces play a role in reducing complement protein binding.     

The interaction between DNA and cationic lipid films at the air-water interface

The interaction between DNA and positively charged dioctadecyldimethylammonium bromide (DODAB) and DODAB/disteroylphosphatidylcholine (DSPC) monolayers at the air-aqueous interface was studied by a combination of the surface film balance and Brewster angle microscopy. In presence of DNA, the Pi-A isotherm of the cationic monolayer shifts to larger mean molecular areas due to the electrostatic interaction with DNA while the typical liquid expanded-liquid condensed phase transition for DODAB monolayers disappear and the monolayer remains to be in the liquid expanded phase. Furthermore, the morphology of the film dramatically changes, where the large dendritic-like condensed aggregates observed for DODAB monolayers vanish. The charge density of the monolayer was varied by using mixed monolayers with the zwitterionic DSPC and no large effect was observed on the interaction with DNA. By modeling the electrostatic interactions with the linearized Poisson-Boltzmann equation using the finite-element method and taking into account the assumption in the dielectric constants of the system, it was possible to corroborate the expansion of the cationic monolayer upon interaction with DNA as well as the fact that DNA does not seem to penetrate into the monolayer.     

Phosvitin-calcium aggregation and organization at the air-water interface

Phosvitin, an egg yolk protein constituted by 50% of phosphorylated serines, presents good emulsifying properties whereas its interfacial properties are not yet clearly elucidated and remain object of discussion. Phosvitin has a high charge density and naturally forms aggregates through phosphocalcic bridges in egg yolk. This high charge density, doubled by this capacity to aggregate, limits the adsorption of the protein at the air-water interface. In this work, we investigated the aggregation impact by calcium ions on the organization of the phosvitin interfacial film using the atomic force microscopy. Phosvitin interfacial films without calcium ions are compared to phosvitin interfacial films formed in the presence of calcium ions in the subphase. We demonstrated that phosvitin is able to anchor at air-water interfaces in spite of its numerous negative charges. In the compression isotherm a transition was observed just before 28 mN/m signifying a possible modification of the interfacial film structure or organization. Calcium ions induce a reorganization towards a greater compaction of the phosvitin interfacial film even at low surface pressure. In conclusion we suggest that, in diluted regime, phosvitin molecules could adsorb by their two hydrophobic extremities exhibiting loops in the aqueous phase, whereas in concentred regime (high interfacial concentration) it would be adsorbed at the interface by only one extremity (brush model).     

Semisynthesis iii - the homoserine 12,105 analogue of hen egg-white lysozyme.

The homoserine ^12,105 analogue of Hen egg-white lysozyme has been prepared by two routes. In the first approach a large excess of cyanogen bromide was used to cleave the native enzyme at methionines 12 and 105, the tertiary structure being maintained by the presence of the four disulphide bonds. Formation of the two new amide bonds was achieved using the activation of the homoserine lactone residues generated at positions 12 and 105; the reaction being most efficient when anhydrous DMSO was used as the solvent. An alternative approach using limited (two-fold excess) cyanogen bromide digestion gave the same homoserine analogue without chain fragmentation. A purified sample of the analogue showed no enzymic activity.     

Structures and rheological properties of hen egg yolk low density lipoprotein layers spread at the air-water interface at pH 3 and 7

Low density lipoproteins (LDL) from egg yolk have a classical structure of lipoprotein with a core of neutral lipids surrounded by a monolayer of apoproteins and phospholipids. This structure collapses during adsorption and all constituents spread at the interface. To understand better the nature of the interactions between apoproteins and lipids at the interface, we have deposited LDL at an air-water interface and analysed the isotherms during their compression on a Langmuir trough. Then, these LDL films were studied by atomic force microscopy (AFM) imaging. To identify the protein and lipid structures, we imaged films before and after lipid solubilisation by butanol. To study the interactions in the LDL films, we have varied the pH, ionic strength and used simplified model systems. We also studied the correlation between observed structures and interfacial rheology of the film. The isotherms of interfacial LDL films were similar for pH 3 and 7, but their structures observed in AFM were different. At surface pressures below the transition corresponding to the demixion of apoprotein-neutral lipid complexes, the LDL film structure was not governed by electrostatic interactions. However, above this surface pressure transition (45mN/m), there was an effect of charge on this structure. Around the transition zone, the rheological properties of LDL films at pH 3 were different as a function of pH (viscous at pH 3 and visco-elastic at pH 7). So, the rheological properties of LDL films could be linked to the structures formed by apoproteins and observed in AFM.     

Hydrogen-bonded monolayers and interdigitated multilayers at the air-water interface

Crystalline monolayers of octadecylsulfonate amphiphiles (C18S) separated by hydrophilic guanidinium (G) spacer molecules were formed at the air-water interface at a surface coverage that was consistent with that expected for a fully condensed monolayer self-assembled by hydrogen bonding between the G ions and the sulfonate groups. The surface pressure-area isotherms reflected reinforcement of this monolayer by hydrogen bonding between the G ions and the sulfonate groups, and grazing incidence X-ray diffraction (GIXD) measurements, performed in-situ at the air-water interface, revealed substantial tilt of the alkyl hydrophobes (t = 49 degrees with respect to the surface normal), which allowed the close packing of the C18 chains needed for a stable crystalline monolayer. This property contrasts with behavior observed previously for monolayers of hexadecylbiphenylsulfonate (C16BPS) and G, which only formed crystallites upon compression, accompanied by ejection of the G ions from the air-water interface. Upon compression to higher surface pressures, GIXD revealed that the highly tilted (G)C18S monolayer crystallites transformed to a self-interdigitated (G)C18S crystalline multilayer accompanied by a new crystalline monolayer phase with slightly tilted alkyl chains and disordered sulfonate headgroups. This transformation was dependent on the rate of compression, suggesting kinetic limitations for the "zipper-like" transformation from the crystalline monolayer to the self-interdigitated (G)C18S crystalline multilayer.     

Interactions at the interface between thermoplastic rubber and polychloroprene adhesive

The interface produced between a chlorinated thermoplastic styrene‐butadiene‐styrene rubber and a polychloroprene (PCP) adhesive has been studied and compared to the interface produced using a polyurethane (PU) adhesive. Chlorination of the rubber was produced by spin coating solutions of trichloroisocyanuric acid in methyl ethyl ketone. The adhesive solution was spin coated on to the chlorinated rubber and the interface between the chlorinated rubber and the adhesive was analyzed by infrared spectroscopy. Chlorination of the rubber produces cross‐linking of the outermost chlorinated and oxidized rubber surface, which becomes insoluble in toluene. The chlorinated rubber chains are able to migrate through the chlorinated rubber/adhesive interface and produce a cross‐linked interface. Similar interfaces are obtained with PU or PCP adhesive. However it is the addition of a thermoreactive phenolic tackifier resin to the PCP adhesive, which imparts appropriate rheological properties to the PCP adhesive, responsible for the increased adhesion properties. Copyright © 2008 John Wiley & Sons, Ltd.     

Aligning nanodiscs at the air-water interface, a neutron reflectivity study

Nanodiscs are self-assembled nanostructures composed of a belt protein and a small patch of lipid bilayer, which can solubilize membrane proteins in a lipid bilayer environment. We present a method for the alignment of a well-defined two-dimensional layer of nanodiscs at the air-water interface by careful design of an insoluble surfactant monolayer at the surface. We used neutron reflectivity to demonstrate the feasibility of this approach and to elucidate the structure of the nanodisc layer. The proof of concept is hereby presented with the use of nanodiscs composed of a mixture of two different lipid (DMPC and DMPG) types to obtain a net overall negative charge of the nanodiscs. We find that the nanodisc layer has a thickness or 40.9 ± 2.6 ? with a surface coverage of 66 ± 4%. This layer is located about 15 ? below a cationic surfactant layer at the air-water interface. The high level of organization within the nanodiscs layer is reflected by a low interfacial roughness (~4.5 ?) found. The use of the nanodisc as a biomimetic model of the cell membrane allows for studies of single membrane proteins isolated in a confined lipid environment. The 2D alignment of nanodiscs could therefore enable studies of high-density layers containing membrane proteins that, in contrast to membrane proteins reconstituted in a continuous lipid bilayer, remain isolated from influences of neighboring membrane proteins within the layer.     

Reorganization of lipid nanocapsules at air-water interface 3. Action of hydrolytic enzymes HLL and pancreatic PLA2

The action of the hydrolytic enzymes humicola lanuginosa lipase (HLL) and pancreatic phospholipase A2 (PLA2) on monolayers formed from lipid nanocapsules (LNC) and model monolayers containing their components, Labrafac, Solutol and Lipoid, is studied by simultaneous measuring the changes in the film area and the surface potential in the "zero order" trough at constant surface pressure (pi). The kinetic models describing the hydrolysis by HLL of the Labrafac, Solutol and their mixtures have been proposed. By using the developed theoretical approach together with the experimental results the surface concentrations of the substrates, hydrolysis products and values of the global kinetic constants were obtained. The comparison between the global kinetic constants in the case of HLL hydrolysis of pure Labrafac, Solutol monolayers and those of the model mixed Labrafac/Solutol monolayers, shows that the rates of hydrolysis are of the same order of magnitude, i.e. an additively of the HLL enzyme action is observed. The composition of the mixed Labrafac/Solutol monolayer, formed after the interfacial LNC destabilization, was estimated.     

Sonication induced sheet formation at the air-water interface

A hydrophobic pentadecapeptide, AGAAAA-GAVVGGLGG (1), part of the prion sequence PrP (106-127), on fresh aqueous dissolution takes a mixture of random and sheet conformations which forms a stable monolayer with a high beta-sheet content when compressed at the air-water interface. This also develops into a kinetically stabilized beta-sheet structure on sonication.     

Transmetalation reaction between hydrophobic silver nanoparticles and aqueous chloroaurate ions at the air-water interface

The transmetalation reaction between a sacrificial nanoparticle and more noble metal ions in solution has emerged as a novel method for creating unique hollow and bimetallic nanostructures. In this report, we investigate the possibility of carrying out the transmetalation reaction between hydrophobic silver nanoparticles assembled and constrained at the air-water interface and subphase gold ions. We observe that facile reduction of the subphase gold ions by the sacrificial silver nanoparticles occurs resulting in the formation of elongated gold nanostructures that appear to cross-link the sacrificial silver particles. This transmetalation reaction may be modulated by the insertion of an electrostatic barrier in the form of an ionizable lipid monolayer between the silver nanoparticles and the aqueous gold ions that impacts the gold nanoparticle assembly. Transmetalation reactions between nanoparticles constrained into a close-packed structure and appropriate metal ions could lead to a new strategy for metallic cross-linking of nanoparticles and generation of coatings with promising optoelectonic behavior.     

Co-adsorption of carboxymethyl-cellulose and cationic surfactants at the air-water interface

We investigated the interaction between an anionic polyelectrolyte (carboxymethylcellulose) and cationic surfactants (DTAB, TTAB, and CTAB) at the air/water interface, using surface tension, ellipsometry, and Brewster angle microscopy techniques. At low surfactant concentration, a synergistic phenomenon is observed due to the co-adsorption of polyelectrolyte/surfactant complexes at the interface, which decreases the surface tension. When the surfactant critical aggregation concentration (cac) is reached, the adsorption saturates and the thickness of the adsorbed monolayer remains constant until another characteristic surfactant concentration, C0, is reached, at which all the polymer charges are bound to surfactant in bulk. Above C0, the absorbed monolayer becomes much thicker, suggesting adsorption of bulk aggregates, which have become more hydrophobic due to charge neutralization.