CIS-01413; No of Pages 17 Advances in Colloid and Interface Science xxx (2014) xxx–xxx

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Langmuir monolayers as models to study processes at membrane surfaces Cristina Stefaniu, Gerald Brezesinski, Helmuth Möhwald ⁎ Max Planck Institute of Colloids and Interfaces, Science Park Potsdam-Golm, Am Mühlenberg 1, D-14476 Potsdam, Germany

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Available online xxxx Keywords: Monolayers Bilayers GIXD IRRAS Interactions Lipids DNA Peptides Nanoparticles Interfacial reactions

a b s t r a c t The use of new sophisticated and highly surface sensitive techniques as synchrotron based X-ray scattering techniques and in-house infrared reflection absorption spectroscopy (IRRAS) has revolutionized the monolayer research. Not only the determination of monolayer structures but also interactions between amphiphilic monolayers at the soft air/liquid interface and molecules dissolved in the subphase are important for many areas in material and life sciences. Monolayers are convenient quasi-two-dimensional model systems. This review focuses on interactions between amphiphilic molecules in binary and ternary mixtures as well as on interfacial interactions with interesting biomolecules dissolved in the subphase. The phase state of monolayers can be easily triggered at constant temperature by increasing the packing density of the lipids by compression. Simultaneously the monolayer structure changes are followed in situ by grazing incidence X-ray diffraction or IRRAS. The interactions can be indirectly determined by the observed structure changes. Additionally, the yield of enzymatic reaction can be quantitatively determined, secondary structures of peptides and proteins can be measured and compared with those observed in bulk. In this way, the influence of a confinement on the structural properties of biomolecules can be determined. The adsorption of DNA can be quantified as well as the competing adsorption of ions at charged interfaces. The influence of modified nanoparticles on model membranes can be clearly determined. In this review, the relevance and utility of Langmuir monolayers as suitable models to study physical and chemical interactions at membrane surfaces are clearly demonstrated. © 2014 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General comparison of monolayers and bilayers . . . . . . . . . . . . . 2.1. Lateral intermolecular interactions . . . . . . . . . . . . . . . . 2.2. Mechanical properties . . . . . . . . . . . . . . . . . . . . . . 2.3. Mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Domain structures . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Grazing incidence X-ray diffraction (GIXD) . . . . . . . . . . . . 3.2. Total reflection X-ray fluorescence (TRXF) . . . . . . . . . . . . . 3.3. Infrared reflection absorption spectroscopy (IRRAS) . . . . . . . . Interactions in and at Langmuir monolayers . . . . . . . . . . . . . . . 4.1. Miscibility behavior studied in two-component Langmuir monolayers 4.2. Biomolecular interactions studied in Langmuir monolayers . . . . . 4.2.1. Lipid–DNA interactions . . . . . . . . . . . . . . . . . 4.2.2. Lipid–peptide interactions . . . . . . . . . . . . . . . . 4.2.3. Lipid–protein interactions . . . . . . . . . . . . . . . . 4.2.4. Lipid–NPs and lipid–polymer interactions . . . . . . . . . 4.2.5. Lipid–synaptic vesicle interactions . . . . . . . . . . . . 4.2.6. Lipid–drug and hormone interactions . . . . . . . . . . . 4.2.7. Lipid–β-cyclodextrin (β-CD) interaction . . . . . . . . .

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⁎ Corresponding author. E-mail address: [email protected] (H. Möhwald).

http://dx.doi.org/10.1016/j.cis.2014.02.013 0001-8686/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Stefaniu C, et al, Langmuir monolayers as models to study processes at membrane surfaces, Adv Colloid Interface Sci (2014), http://dx.doi.org/10.1016/j.cis.2014.02.013

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4.3. 4.4. 4.5.

Ions at Langmuir monolayers . . . . . . . . . . Chemical reactions in Langmuir monolayers . . . Langmuir monolayers as templates . . . . . . . 4.5.1. Biomineralization . . . . . . . . . . . 4.5.2. Construction of functional nano-materials 5. Phospholipid bilayers at the solid–liquid interface . . . . 6. Conclusions and outlook . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Since the early days of the membrane model of Singer and Nicholson [1], phospholipid monolayers have been considered good models of biomembranes, as they represent half of a membrane (Fig. 1). But the early work with amphiphile monolayers was largely hampered by the absence of tools to investigate liquid interfaces with molecular and microscopic resolution. This has changed drastically in the last 30 years since many interface sensitive techniques have become applicable to fluid interfaces (X-ray [2,3] and Neutron scattering [4,5], ellipsometry [6], fluorescence [7] and Brewster angle microscopy [8,9], Reflection–absorption FTIR-spectroscopy [10], nonlinear optical spectroscopy [11]). Therefore studies of Langmuir monolayers have encountered a drastic increase in activity, although they are not directly relevant for any applications. Yet, due to their high definition they are excellent models for many areas with application potential. They are the precursors for Langmuir–Blodgett films [12,13], organized monolayers possessing potential for coatings and organic electronics. They present the interfaces in emulsions, a basic structure in colloid science. Here we concentrate on their value as 2D models of biological membranes (phospholipid monolayers at the air/water interface). In the first part we will focus on general aspects, comparing monolayers and bilayers, whereas in the second part we will present modern examples on their real strengths, studies of interactions at the membrane surface. One major difference between monolayers and bilayers concerns thermodynamics. Condensed phases of monolayers are in many cases metastable states whereas bilayers in multilamellar systems represent thermodynamically stable states. Moreover, the lipid polymorphism is richer in liquid crystals in three-dimensional systems than in 2D systems. The 3D structure of a collapsed monolayer is less hydrated than the corresponding fully hydrated bilayer in the LC phase [14]. The dynamics within monolayers and bilayers constitutes an additional

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difference. As concerns the structure, the recent works of Ziblat et al. [15] showed that the interactions between leaflets of ceramide/ cholesterol and phospholipid/cholesterol systems led to phase separation, changes in molecular tilt angle or formation of cholesterol crystals. The structures are similar but the unit cell dimensions depend on the number of juxtaposed layers (monolayers, single hydrated bilayers, and multilayers). The in-plane fine structure in multilayers and monolayers of lipid mixtures is in many cases different (not the tilt angle of the chains but the direction and rate of lattice distortion) [16]. As a consequence, employing one or the other physical model to describe real biological membranes could give different information regarding in-plane structures and the influence of other biological molecules as peptides or proteins on lipid structures. In this review, we concentrate on results obtained in the last years with 2D model systems (Langmuir monolayers at the soft air/liquid interface). Our intention is to highlight the usefulness of monolayers in studies of interfacial interactions in model systems well suited to answer important questions in material and life sciences. 2. General comparison of monolayers and bilayers 2.1. Lateral intermolecular interactions Inspecting the cartoons in Fig. 1 it is obvious that the lateral interactions in monolayers and bilayers are similar, and therefore also theoretical models are comparable. In the bilayer, the neighboring monolayer is expected to provide a weak additional interaction, and for the case of the biomembrane, where the membrane is fluid, this may be approximated by an oil with very low interfacial energy. Of course the monolayer may undergo drastic changes in density and hence exhibit phase transitions from gaseous to condensed phases [17], whereas the bilayer does not exhibit gaseous phases but only

Fig. 1. Representation of a biological membrane as a multicomponent system (left). The two most used models are: Aqueous dispersions of lipid bilayers (right bottom) and planar lipid monolayers on an aqueous subphase (right top).

Please cite this article as: Stefaniu C, et al, Langmuir monolayers as models to study processes at membrane surfaces, Adv Colloid Interface Sci (2014), http://dx.doi.org/10.1016/j.cis.2014.02.013

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condensed (liquid-crystalline and gel) ones. Therefore here we concentrate on transitions between condensed (fluid and solid) phases and only on lamellar phases in vesicle dispersion despite the fact that such dispersions exhibit many 3D phases [18]. Most pronounced is the first-order phase transition from a liquidcrystalline (fluid) to a gel (ordered) phase of lipid bilayers which can be compared to a main phase transition distinguished by a break in the slope in pressure/area diagrams of lipid monolayers. There has been a long debate on the nature of this transition in view of the fact that a horizontal isotherm slope has never been observed [19]. In most cases residual impurities may explain this slope and there is largely consensus that there is a first-order phase transition [20]. Different from most atomic or molecular systems the entropy of this transition is not determined by freezing in lateral motions (although this exists) but by the ordering of the aliphatic chains: because of the low energy of gtg-kinks and others, the chain alignment in the liquid phase exhibits many of these defects [21], and the transition into the ordered phase involves the removal of these defects. In line with this, transition entropy and enthalpy scale linearly with chain length, and this also concerns transition temperatures or pressures [22]. This holds for monolayers as well as for bilayers and similarly the influence of interactions in the head group region of lamellar phases with enough excess of water can also be explained. In this case, Coulomb interactions can be screened, removed or increased by salt, multivalent ions or pH, and again transition temperatures and pressures can be varied as theoretically predicted [23]. The lamellar phases of lipid multilayers have been frequently mapped on those of smectic liquid crystals that are distinguished by different degrees of order within and normal to a plane. This mapping as regards in-plane ordering has been also possible for monolayers, and especially detailed studies have been performed with single-chain molecules. A zoo of ordered states has been discovered, predominantly by grazing incidence X-ray diffraction (GIXD) [24]. So-called rotator or hindered rotator phases were detected where the chains are in all-trans configuration and may partly rotate about their long axes (Fig. 2). These long axes may form a distorted (or undistorted) hexagonal lattice with short correlation length and be tilted towards nearest or next-nearest neighbors or be untilted. At lowest temperature or longer chain lengths there are various crystalline phases. These crystalline phases possess long-range order and a small chain cross section (18.0–18.2 Å2), the rotator phases are distinguished by cross sections between 19.5 Å2 and 20.5 Å2,

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and upon compression the lattice parameters vary continuously within one phase [25]. The question now arises how this picture changes if, like in most phospholipids, two aliphatic chains are coupled by a head group? Although a chain rotation is then not possible, one measures chain cross sections and diffraction line widths characteristic for rotator or hindered rotator phases [26,27]. Apparently dynamic disorder has been converted into a static one. Still chain tilt into different lattice directions has been observed, and the tilt angle is a sensitive indicator of lateral interactions as will be shown below. Physically there is another qualitative difference for double-chain compared to single-chain systems: The centers of the head groups could be ordered causing a doubling of the unit cell. This has been observed for phospholipid crystals but only rarely for bilayer phases at high water content [28]. The appearance of so-called subgel phases with the ordering of entire molecules observed in phospholipid dispersion is connected with a partial dehydration of the head group during long incubation times at low temperatures [29,30]. But in most cases the head groups are positionally not ordered in bilayers as well as in monolayers. However, for enantiomerically pure monolayers the inplane lattice exhibits an oblique distortion of the hexagonal symmetry [26]. This indicates an orientational order of the head groups possessing a chiral center. It is interesting to note that only recently a monolayer phase with a supercell composed of three molecules of a GPI fragment has been observed [31]. The observed monolayer structure is reminiscent of subgel phase structures observed in lipid dispersions. The head group ordering is observed regardless of any incubation period, because of a strong hydrogen bond network which rigidifies the monolayer structure. 2.2. Mechanical properties The bending elasticity, a parameter so important in the brilliant work of W. Helfrich, is of very different origin in comparing monolayer and bilayer [32]. For monolayers in liquid-crystalline phases it is dominated by the surface tension which is negligible for bilayers. Only for crystalline films the bending elasticity is a directly phase-related property that can be deduced from X-ray reflectivity measurements [33]. From X-ray reflectivity studies with monolayers one could also deduce three-dimensional density changes with pressure or temperature. Typically the corresponding elastic moduli or expansion coefficients are much higher for monolayers compared to bilayers. This can be explained by the fact that upon monolayer expansion there may be free volume, whereas upon bilayer expansion a neighboring alkane segment may fill the space. 2.3. Mixtures

Fig. 2. Generalized phase diagram of Langmuir monolayers of single-chain amphiphiles. There are first-order (solid lines) and second-order (dashed lines) phase transitions between the different condensed phases. At low lateral pressure π, the molecules are tilted, at high compression the molecules are untilted. Lowering the temperature leads to improved packing going from rotator phases with larger cross-sectional areas via 1D to 2D crystalline phases with herringbone or pseudo-herringbone packing modes and small cross-sectional areas.

As biological membranes consist of complex mixtures, miscibility has been an important issue. There are classical rules in the field of liquid crystals on the immiscibility of molecules in different phases, and these hold for monolayers as well as for bilayers [34]. However, the miscibility of lipids from different classes has been found to differ in monolayers and bilayers depending on the polar head groups. Most interesting, also from a biophysical point of view, are the phases involving cholesterol in mole-fractions above 20%. There coexist liquid phases with high and low cholesterol content [35]. They exhibit different dynamics, compressibility and solvent potency for other lipids and proteins and are often discussed in connection with lipid rafts [36]. A qualitative difference of monolayers and bilayers concerns the chain length dependence of miscibility. It is known that molecules differing in chain length by more than 4 CH2 groups are not completely miscible in bilayers and this is reasoned by a mismatch at the interface between the leaflets causing high elastic energy [37,38]. This would not exist in a monolayer, but has not been proven to the best of our knowledge.

Please cite this article as: Stefaniu C, et al, Langmuir monolayers as models to study processes at membrane surfaces, Adv Colloid Interface Sci (2014), http://dx.doi.org/10.1016/j.cis.2014.02.013

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2.4. Domain structures

3.1. Grazing incidence X-ray diffraction (GIXD)

In the early 70's it was already concluded from electron spin resonance and electron microscopic studies, that there are domains of ordered phases in a lake of disordered phase of bilayers [39], and these domains later became popular as ‘rafts’ [40,41]. Such domains with dimensions of some hundred molecules were also supposed to exist in monolayers [42], and they could also explain the finite slope in the phase coexistence range of the pressure/area isotherms [43] of lipid monolayers. All the more it was surprising that domains could be observed by fluorescence microscopy [7,44], but these domains had sizes of millions of molecules. They were stained by dyes with preferential solubility in one of the coexisting phases and, although dye concentrations could be kept below 1 mol%, there was some doubt on the dye influence. However later developed Brewster angle microscopy [8,9] made these domains also visible without using any staining dye. Following these monolayer studies there was an intense search for domains in bilayers by fluorescence microscopy, but it took almost 20 years until these were found [45,46]. The coexisting phases could be 2 fluids with different composition or solids and fluids, and these are meanwhile observed for monolayers as well as for bilayers. For monolayers the remarkable domain shapes and superlattices found have led into an entirely new field of physics in 2 dimensions:

There are only a few beamlines at synchrotron sources worldwide allowing to study the ordering of molecules in a periodic twodimensional array in monomolecular films at the air/liquid interface. GIXD is not only the method of choice for the determination of the molecular organization (unit cell dimensions) but also of the orientation of molecules with respect to the interface (tilt and tilt direction). A conventional Langmuir trough is placed in a helium-flushed container with capton windows transparent for X-rays. The monochromatic X-ray beam is adjusted to strike the liquid surface at an angle of incidence slightly below the critical angle for total reflection in order to produce an evanescent wave, which propagates along the surface and can be diffracted by lateral structures in the monolayer. The diffracted intensity can be monitored by a linear position sensitive detector (PSD) as a function of the vertical scattering angle αf and the horizontal scattering angle 2θxy. The scattering vector Q is written in terms of an in-plane component Q xy depending on the horizontal scattering angle 2θxy and an out-of-plane component Q z depending on the vertical scattering angle αf. Analogous to 3D systems, Bragg peaks are indexed by Miller indices h and k. Lattice spacings are given by dhk ¼ 2π hk and related to the lattice parameters a and b. From the in-plane and

1) The confinement of molecular transport within the surface enabled the study of crystal growth in 2D, while heat can escape into the 3rd dimension [47,48]. 2) Molecules at interfaces possess dipole moments that are oriented with respect to the normal or that disturb the orientation of the water dipoles [49,50]. Therefore domains of one phase exhibit an excess dipole density in normal direction, so that these two domains repel each other [51]. Summing up the interaction energy of a dipolar disk yields a logarithmic divergence of the repulsive energy with domain diameter [52]. This causes a finite domain size and hence a superlattice known as Wigner lattice. Conceptually the interplay between electrostatic energy and line tension can also cause a deviation from circular domain shapes, and in fact many shape transitions have been observed that could be introduced via pH, temperature, salt or composition [44,53]. Concerning the latter, cholesterol plays an interesting role by causing elongated domain shapes [54]. Presumably it is enriched at domain boundaries thus reducing the line tension even if its content is only some mol%. The most interesting shapes are the elongated ones exhibiting chirality, and these typically occur if an underlying lattice is not hexagonal but rectangular (distorted hexagonal). Then the line tension is also not isotropic, and an impurity like cholesterol may preferentially influence specific crystal faces reducing their energy [25]. If in addition the head groups are arranged according to their chirality, domain elongation may follow this resulting in chiral domain shapes. For symmetric bilayers one does not expect these repulsive dipolar interactions. For aligned head groups or for uniform chain tilt there may be in-plane dipolar interactions, but these should be attractive, not limiting the domain size or causing a superlattice. Hence for a finite domain size in equilibrium one has to invoke other forces, and elastic interactions are most probable candidates. Consequently there exist micron sized domains in monolayers as well as in bilayers, but at least in symmetric bilayers the underlying forces must be different. 3. Methods Synchrotron based X-ray scattering techniques and in-house infrared reflection absorption spectroscopy (IRRAS) are some of the most important methods to study monolayer structures and interactions between lipid monolayers and molecules dissolved in the subphase. Here, we will only shortly describe some basics of these important modern methods.

Q xy

out-of-plane peak positions one derives information about the tilt angle and the tilt direction. Model peaks taken as Lorentzian in the in-plane direction and as Gaussian in the out-of-plane direction are leastsquare fitted to the observed intensities [25,55–60]. 3.2. Total reflection X-ray fluorescence (TRXF) In the last years, TRXF has been developed as a simple and quantitative analytical method for the characterization of, e.g., the ionization state of molecules at the liquid/air interface or ion competition in the electrical double layer (Hofmeister series) [61–63]. The monochromatic X-ray beam strikes the liquid surface at grazing incidence to be highly surface sensitive (penetration depth of ~8 nm). The used X-ray energy depends on the type of the element to be detected. The fluorescence signal can be measured by an energy sensitive detector with an entrance window placed either parallel or under a certain angle to the liquid surface. The fluorescence intensity of an element depends only on the experimental conditions. Therefore, a simple calibration procedure can be applied using monolayers with known charge densities on subphases containing only one type of counter-ion. 3.3. Infrared reflection absorption spectroscopy (IRRAS) IR spectra can be recorded by using FTIR spectrometers attached to an external air/liquid reflection unit. The IR beam is conducted out of the spectrometer and focused onto the liquid surface of a temperature controlled Langmuir trough. The measurements can be carried out with p- and s-polarized light at different angles of incidence above and below the Brewster angle. Measurements are performed by using a trough with two compartments. One compartment contains the monolayer system under investigation (sample), whereas the other is filled with the pure subphase (reference). The trough is shuttled so that the IR beam illuminates either the sample or the reference. The single-beam reflectance spectrum from the reference trough is taken as background for the single-beam reflectance spectrum of the monolayer in the sample trough to calculate the reflection–absorption spectrum as -log(R/R0) in order to eliminate the water vapor signal [64–68]. IRRA spectra can be simulated on the basis of the optical model of Kuzmin and Michailov [69,70]. The intensity and shape of a reflection absorption band depend on the absorption coefficient, the full-width at half-height, the orientation of the transition dipole moment within the molecule, the molecular tilt angle, the polarization and the angle of incidence of the incoming light, as well as on the

Please cite this article as: Stefaniu C, et al, Langmuir monolayers as models to study processes at membrane surfaces, Adv Colloid Interface Sci (2014), http://dx.doi.org/10.1016/j.cis.2014.02.013

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layer thickness and its refractive index. Simulated spectra can be fitted to the experimental data as follows: In a first step, the layer thickness and the refractive index are determined from a fit of the OH stretching vibrational band in the range of 3800–3000 cm−1. With these parameters taken as constants, other vibrational bands can be fitted to yield information about the orientation of the molecules. 4. Interactions in and at Langmuir monolayers In the last years, Langmuir monolayers have been used as extremely versatile and easy to handle model systems allowing the investigation of panoply of interactions with different species of compounds cospread at the interface or dissolved in the subphase. Amphiphilic molecules forming the monolayers are trapped at the air/water interface and give indirect information about interactions with the other components (ions, drugs, DNA, peptides, polymers, NPs, etc.) via changes detected by highly surface sensitive techniques as GIXD, X-ray reflectivity (XR), total reflection X-ray fluorescence (TRXF) and IRRAS. 4.1. Miscibility behavior studied in two-component Langmuir monolayers Two-component Langmuir monolayers, obtained by co-spreading of two amphiphiles as mixtures in an appropriate organic solvent at the air/water interface, have been intensively used for gaining a better understanding of their molecular interactions. In case, the single components form condensed monolayers, GIXD data allow the identification of their miscibility behavior with Angstrom resolution and the elaboration of two-dimensional phase diagrams. This is the first step in the direction of better models for biological membranes as multicomponent systems. The conclusions made from the analysis of pressure/area isotherms regarding miscibility or demixing can be sometimes misleading. E.g., a linear relation between molecular area and mole fraction shows either an ideal miscibility or a complete demixing. In contrast, monolayer structures can be determined for singlecomponent films as well as for binary mixtures by GIXD and lead to unambiguous phase diagrams. Thus, an ideal miscibility was found for mixed monolayers of two chemically similar amphiphiles HTPA (3-hydroxy-N-tridecyl propanoic acid amide) and TDAHA (tetradecanoic acid-(2-hydroxyethyl)amide) [71]. The molecular tilt angle passes through a minimum at equimolar mixture. This was connected to a maximum of the cross-sectional area and a minimum in the entropy change during the LE/LC phase transition. The observed phase diagram of the mixed TDAHA-HTPA monolayers shows the existence of a large composition range in which a NNN (next nearest neighbor) tilted orthorhombic phase is enclosed between the oblique phases of the pure compounds (Fig. 3). Such a behavior was connected to the ability of the head groups to form hydrogen bonds. The study was extended by additionally adding into the system the corresponding N,O-diacyl derivative of ethanolamine tetradecanoic acid-2-[(1-oxotetradecyl) amino]ethyl ester (C13H27–CO–NH–(CH2)2– O–CO–C 13H27 , TAOAE) [72]. The main characteristics of onecomponent Langmuir monolayers (the morphology of the LC phases, the two-dimensional lattice structures) of acid amide amphiphiles with one or two alkyl chains are fundamentally different. The binary mixtures (TDAHA/TAOAE, HTPA/TAOAE (1:1)) as well as the ternary ones (TAOAE/TDAHA/HTPA (1:1:1)) formed homogeneous mixed Langmuir monolayers, with a strong dominance of the double-chain compound (TAOAE) as unequivocally proven both at micrometer (BAM) and Angstrom scales (GIXD) [72]. In another interfacial study, the miscibility behavior of oppositely charged molecules was studied. The equimolar monolayer of stearic acid (SA) and stearylamine (ST) is effectively uncharged and characterized by a homogeneous structure showing complete miscibility of the components [73]. TRXF was used to quantify the binding of Ca2+ and Cs+ to the SA monolayers and of I− and Cl− to the ST monolayers,

Fig. 3. Pressure versus mole fraction of TDAHA phase diagram measured at 5 °C. LE denotes the fluid phase, obl 1 is the oblique phase with a cross-sectional area of ~19.3 Å2 and obl 2 the one with the larger cross-sectional area (~20.0 Å2) found in HTPA monolayers, the orthorhombic phase has a NNN tilt, obl 3 is the oblique phase of TDAHA. The corresponding contour plots (corrected diffraction intensities as a function of the inplane Qxy and out-of-plane Qz components of the scattering vector) of the different phases are shown. Figure adapted from [71].

while no binding of these ions could be detected for the mixed SA/ST films [73]. Interestingly, chiral textures have been identified inside twodimensional achiral domains formed in Langmuir monolayers of mixtures (equimolar ratio) of the anionic phospholipid dimyristoyl phosphatidic acid (DMPA) and the cationic amphiphilic derivative of the hemicyanine dye (HSP) [74]. This phenomenon was attributed to the ordered self-aggregation of the polar hemicyanine groups which were able to rotate for reducing the repulsion between dipole moments. The authors considered that the phenomenon might be regarded as a chiral transfer from the chiral center of DMPA to the chromophore [74]. Additionally, self-assembly and molecular recognition have been observed in Langmuir monolayers of equimolar mixtures of adenine- and thymine-functionalized nucleolipids (9-(2-octadecyloxycarbonylethyl) adenine and 1-(2-octadecyloxycarbonylethyl)thymine formed at the air/water interface [75]. Based on surface pressure–molecular area isotherms and IRRAS measurements, the study highlights that on aqueous subphases containing complementary bases no significant molecular recognition was observed for monolayers of individual nucleolipids. Opposite this, in monolayers of an equimolar mixture molecular recognition occurred between the adenine and thymine moieties through hydrogen bonding probably with the development of cyclic structures of adenine–thymine–adenine–thymine quartets [75]. Moreover, for mimicking and understanding the intermolecular interactions in bacterial membranes different mixtures of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) have been investigated in Langmuir layers. The results defined a non-ideal mixing behavior, the most favorable packing of molecules occurring at equimolar composition [76]. This was explained by formation of hydrogen bonds between the PE and PG head groups. Complete miscibility has been observed for binary mixtures of a biological essential single-chain ether phospholipid PAF (1-O-Octadecyl-2acetyl-sn-glycero-3-phosphocholine) [77] with DPPC and cholesterol when studied in Langmuir monolayers. The strong interactions of PAF with cholesterol were in contrast with the thermodynamically unfavorable interactions of PAF with DPPC. Another interfacial study highlighted the segregation observed in binary Langmuir layers of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE) and cholesterol [78]. Thus, GIXD clearly reflected that at low concentration

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of cholesterol (10 mol%) the monolayer structure of SOPE was disturbed while at higher concentrations (33, 50, and 67 mol%) the formation of sterol-poor and sterol-rich domains occurred [78]. A recent monolayer study revealed the concentration dependent mixing/demixing behavior of a glycosylphosphatidylinositol (GPI) fragment (GlcNα → 6myoIno-1-phosphodistearoyl-glycerol) with a typical low-melting model membrane phospholipid POPC (1-palmitoyl-2oleoyl-phosphatidylcholine) [31]. Below a certain threshold concentration, the GPI-fragment proved to be miscible with POPC, inducing order into the liquid-disordered POPC phase, while above the threshold concentration the GPI-fragment phase-separated due to the strong head group interactions. This behavior was related to the existence of liquid-ordered domains (rafts) in cell membranes with possible answers to the intermolecular interactions of free GPIs or proteinanchored GPIs in real cell membranes. 4.2. Biomolecular interactions studied in Langmuir monolayers 4.2.1. Lipid–DNA interactions Langmuir monolayers are valuable tools for studying the coupling of DNA to cationic lipids. Monitoring the lipid/DNA interaction is a necessary key step for the development of DNA-based pharmaceuticals for gene therapy, biosensors and nano-devices. In this line, to obtain more effective and safer DNA vectors, new cationic lipids are continuously synthesized. Here we describe as one example the phase behavior as well as the interactions with DNA of two new cationic lipids: 2tetradecylhexadecanoic acid-2-[(2-aminoethyl)amino]ethyl-onamide

(CI) and 2-tetradecylhexadecanoic acid-2-[bis(2-aminoethyl)amino] ethylamide (CII) [79]. The changes produced upon interaction with DNA were followed using film balance measurements, GIXD and XR. The results revealed that the subphase pH value (acidic or alkaline) influenced the physical–chemical properties of the monolayers by changing the protonation degree of the head group. Surprisingly, DNA coupling generated the same one-dimensional periodicity of ordered DNA strands adsorbed at the lipid monolayer, independent of the pH value of the subphase (Fig. 4). The study additionally revealed that lipids having a branched polyamine head group structure (CII) seem to interact stronger with the DNA. These results proved to be in agreement with the good transfection efficacy and the low in vitro toxicity, indicating that lipids with such structures are promising candidates for the development of successful non-viral gene delivery systems [79]. Since their pK values at the interface were unknown, an additional TRXF study was employed to reveal the protonation degree of the above-described cationic lipids in monolayers formed on different pH subphases [62]. A simplified TRXF method was employed to determine the concentration of bromide counterions bound to the positively charged lipid head groups. This fluorescence intensity can be directly translated into charge density based on the X-ray fluorescence intensity measured for dioctadecyldimethylammonium bromide monolayers with a known charge density as reference. The results clearly show that monolayers of N-2-{[bis-(2-aminoethyl)amino]ethyl}-2,N′dihexadecyl-propandiamide (CIII) are deprotonated above pH values of 9, while the highest protonation is registered on pH 3 solutions (Fig. 5). The study establishes that the protonation rate depends not

Fig. 4. The chemical structure of compound CII is shown above. (A) Molecular model of DNA ordering at the cationic lipid monolayers. (B) Contour plot of the corrected X-ray intensities as a function of the in-plane (Qxy) and the out-of-plane (Qz) scattering vector components showing the 1D periodicity in an adsorbed DNA layer. (C) The lattice spacing between aligned DNA strands (dDNA) as a function of the surface pressure π. The monolayer of compound CII is formed on a subphase containing 0.1 mM DNA in citric buffer, pH 4 (Δ) or in Tris buffer, pH 8 (●). Figure adapted from [79].

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Fig. 5. Selected X-ray fluorescence spectra of a Langmuir monolayer of compound CIII (chemical structure is inserted) at 40 Å2·molecule−1 on Br−containing subphases at pH 3 ( ), pH 6 ( ), pH 8 ( ) and pH 11 (●) showing the dependence of the protonation state of the head group on the pH of the subphase. Figure adapted from [62].

only on the head group structure but also on the monolayer phase state (packing density). As an example, the protonation degree of the fluid monolayer was estimated to be 1.4, whereas only 1 positive charge per molecule was found in the case of a condensed monolayer of a compound with the same head group structure but different chain pattern [62]. In a similar study, monolayers of N′-2-[(2,6-diamino-1-oxohexyl) amino]ethyl-2,N-(dihexadecyl)propane diamide (lipid 7) and N′-2[(2,6-diamino-1-oxohexyl)amino]ethyl-2-hexadecyl-N-[(9Z)-octadec9-enyl]propane diamide (lipid 8) were studied at the air/water interface as pure one-component and bi-component lipid/DNA systems. These compounds belong to a series of 9 new structural similar lipids designed and synthesized for gene transfection [80]. Since the lipids had the same head group structure but a differed hydrophobic part, the study focused on identifying the influence of the lipid phase state on their ability to bind DNA [81]. Thus, the GIXD data revealed that lipid 7 with two saturated alkyl chains was able to form condensed monolayers (Fig. 6). An orthorhombic lattice with slightly tilted chains characterized the monolayer structure at pH 4 with quite large cross-sectional areas per chain (A0) due to strong electrostatic repulsion between the charged head groups, while at pH 8 (mostly unprotonated head groups) a typical herring-bone arrangement was measured. An additional low intensity peak was detected at Qxy = 1.32 Å−1 (d = 4.76 Å) and Qz = 0 Å−1 for both pH values and assigned to the formation of a hydrogen bond network by the lipid head groups. Considered as a typical peak for hydrogen bonds (N\H···O_C) encountered in β-sheets of proteins and peptides, the study highlights the very first proof of such a strong H-bond network in lipid monolayers. Upon interaction with DNA important changes of the monolayer structure have been recorded. Thus, the peaks corresponding to the H-bond formation vanished completely at pH 4 and had a weak intensity for pH 8 subphases. This fact was attributed to DNA binding to the head groups, disturbing thus the H-bond network. Concerning the monolayer structure, no order could be detected at low pressures at pH 4 suggesting that the coupled DNA fluidized the lipid monolayer, while at higher surface pressures (20 mN/m) the broad Bragg peak measured indicates a hexagonal packing of poorly correlated chains. Interestingly, the DNA binding at pH 8 affected the monolayer structure in a different way. Four strongly overlapping diffraction peaks suggest the coexistence of two phases: one phase of the pure lipid and another phase of the lipid bound to DNA, characterized by larger A0 values and a change of the tilt direction from NN to NNN [81]. Lipid 8 with one double bond in the chain region formed only monolayers in a liquid-disordered state on both studied subphases (pH 4 and 8). The amount of DNA bound to the monolayers was dependent on the lipid charge density. Surprisingly, even at pH 8 (mostly unprotonated state) both lipids bind considerable amounts of

Fig. 6. The chemical structure of lipid 7 is shown above. (A) Schematic illustration of the DNA binding to a compressed (20 mN/m) lipid monolayer. DNA is located underneath the lipid layer and partly penetrated into the head group region. A certain part of the strands orders with a 1D periodicity, another part is disordered. The lipid layer has a high packing density and therefore high charge density. Subsequently, DNA packs also tightly and parts of the macromolecules are extended into the subphase (sterical reasons). (B–E) Representative contour plots of the lipid on different subphases (B – buffer, pH 8, 30 mN/m, C – DNA solution, pH 8, 30 mN/m, D – buffer, pH 4, 30 mN/m, E – DNA solution, pH 4, 30 mN/m) at 20 °C. Figure adapted from [81].

DNA, probably due to an increased protonation degree of the monolayer induced by the approaching polyelectrolyte DNA. The interaxial repeat distance of DNA strands could be measured as well, and it again proved to be strongly dependent on the phase state of the lipids. While the compression of a liquid-like monolayer led to a strong decrease of dDNA, the DNA repeat distances did not considerably change upon compression of condensed monolayers. At the monolayer lift-off point, the charge density in the condensed lipid monolayer is already much higher than the charge density of aligned DNA. In this case, DNA is electrostatically attached to a highly charged plane and not to single lipid molecules. Even if the used transfection vectors are composed of liposomes complexed with DNA, the DNA quantification is another necessary key element for the development of efficient transfection systems. Along this line two extremely valuable methods have been recently established for the quantification of DNA bound to positively charged Langmuir monolayers: IRRAS for phosphate group free lipids and TRXF for bromine labeled DNA (Br-DNA). Having a detection limit of 10–20 μg, the TRXF analytical procedure consists of using bromine as a marker (due to its high absorption cross

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compressed on a subphase containing the complementary mononucleotide guanosine or hybridized with complementary ssDNA strands in the subphase, suggested the appearance of enantiomeric structures, capable of enantioselective binding of their natural ligand, guanosine, solely as a result of surface induced asymmetry in ‘left’ but not in ‘right’ form [82].

Fig. 7. TRXF spectra of DODAB at 30 mN/m on a subphase containing 0.1 mM Br-DNA and different concentrations of KCl at 20 °C: black line — 1 mM KCl; orange line — 3 mM KCl; red line — 10 mM KCl; blue line — 100 mM KCl; green line — 100 mM KCl subphase without DODAB. Figure adapted from [62]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

section and strong K emission line) for quantifying the amount of bromine and consequently the amount of DNA adsorbed to the lipid monolayer (Fig. 7) [62]. Even solutions contaminated with bromide anions can be used since the Br-DNA macromolecules win the adsorption competition with singly charged bromide anions due to the high negative charge. Extremely important for further applications of novel cationic lipids as transfection tools is the fact that the amount of adsorbed Br-DNA increases with increasing electrolyte concentration up to physiological values. This finding was explained by the strong interactions of the anions with the macromolecule resulting in a reduction of the effective charge which requires larger amounts of Br-DNA for the compensation of the overall monolayer charge [62]. The other important method to determine the amount of DNA adsorbed to non-phospholipid monolayers is IRRAS [81]. Typical DNA − bands as the asymmetric, νas(PO− 2 ), and symmetric, νs(PO2 ), phosphate diester stretching bands at 1250–1220 cm− 1 and 1086–1072 cm− 1, respectively, as well as the DNA backbone band at 970 cm− 1 have been used to quantify and to compare the relative amounts of DNA of one sample at different pressures or even of different samples. Significant differences have been found for lipids at pH 4 in comparison to pH 8, if the absolute amount of DNA at the surface is considered. The high charge density at pH 4 resulting from the protonation of the head groups leads to strong electrostatic interaction with the negatively charged macromolecules. Interestingly, the used lipids were able to bind almost the same amount of DNA at the same charge density (same area per molecule) independent of the phase state of the lipid monolayer. In contrast, the electrostatic interactions are reduced at pH 8 due to deprotonation of the head groups. Therefore, a much smaller amount of DNA is adsorbed than at pH 4, but again higher than expected for an almost deprotonated state. The reason is the same as discussed above. The findings of another Langmuir monolayer study were related to the intriguing question of chiral selection during the early period of the ‘Origin of Life’ [82]. The authors show that by irradiating achiral compounds with circularly polarized light, chiral surface structures can be formed which lead to the amplification of biopolymer binding of particular handedness. Thus, mixed Langmuir films of polyconjugated polydiacetylene (PDA) derivatized with cytosine (10,12-pentacosadiynecytidyl, PDC) monomers and alcohol-terminated diacetylene lipid (10,12pentacosadiynol, PDOH) in a 3:1 ratio were formed and polymerized with circularly polarized light (CPL) or non-polarized UV light. The observed difference between left- and right-CPL polymerized films,

4.2.2. Lipid–peptide interactions Accepted as simple 2D membrane models, the phospholipid monolayers have been intensively used in the last years for physical–chemical studies of peptide–membrane interactions. Generally, uncharged phosphatidylcholines (PCs), sphingomyelines (SMs), and cholesterol are often used to model the eukaryotic cell membrane, whereas negatively charged phosphatidylglycerols (PGs), cardiolipin, as well as mixtures of these lipids have been used to mimic the cytoplasmic bacterial membrane [83]. One example is the interaction between amphipathic antimicrobial peptides (AMPs) and phospholipid monolayers. Considered as highly selective, the AMPs are interesting lead structures for the development of new drugs, complementing or even replacing the standard antibiotic therapies. Aiming to understand the mechanism of interaction between AMPs and cellular membranes, a model peptide, the arenicin-1 (Ar-1), which possesses antibacterial and antifungal activities was studied in interaction with phospholipid Langmuir monolayers [84]. The Ar-1 is a natural cyclic peptide of 21 amino acid residues, 6 of which are positively charged arginines and 2 (sulfur-containing) cysteines causing the large 18-residue portion to loop, folding into a unique twisted betasheet structure [85]. This peptide has been compared with the linear derivative (C/S-Ar-1) having serine residues instead of cysteines. Thus, GIXD and IRRAS experiments revealed that both original and modified arenicins strongly interact with negatively charged phospholipids (DPPG). These interactions induce disorder (fluidization of the phospholipid alkyl chains) in the lipid monolayer structure. In contrast, the influence of these AMPs on zwitterionic phospholipids (DPPC) is extremely weak. Moreover, beyond surface pressures of 30 mN/m, considered as the internal pressure of lipid bilayers, both peptides were squeezed out from zwitterionic lipid monolayers, but remained still attached and partly incorporated into anionic lipid monolayers. Thus, the study identified a membrane destructive mechanism (fluidization of the lipid layer) of the peptides, stressing the interplay between hydrophobic and electrostatic interactions involved in the process [84]. Another model membrane composed of 90:10 mol% DPPC:DPPS (dipalmitoyl phosphatidylcholine:dipalmitoyl phosphatidylserine) was used for studying the interaction with purothionins at the air/water interface [86]. As low molecular weight (~ 5 kDa) polypeptides, the purothionins are plant toxins considered to act by lysing the membrane of pathogenic organisms, yet the interaction mechanism was not clarified. Thus, XR and GIXD revealed indeed that the protein disturbs the lateral packing of the phospholipids and increases the fraction of the liquid phase in the monolayer. Interestingly, the data indicated that the protein was bound only for a short time (4 h) to the monolayer after which, by partial withdrawing DPPS molecules from the interface, the monolayer structure was proved to be reminiscent of pure DPPC monolayers. So, the obtained results supported the solubilization mechanism [86]. The ion-dependent interaction of monolayers of lipopolysaccharides from Salmonella enterica rough strains R90 (LPS Ra) with natural and synthetic peptides was recently reported [87]. The electron density profile and the ion distribution near the interface have been determined by XR and TRXF. Thus, in the absence of Ca2+, the natural fish protamine was able to incorporate deeper and to disrupt the stratified lipid structure, whereas in the presence of Ca2 + the peptide was not able to penetrate the more rigid layer. In contrast, the synthetic antisepsis peptide Pep 19–2.5 incorporated into the monolayer in the vicinity of the uncharged sugar units both in the presence and absence of Ca2+ ions [87].

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4.2.3. Lipid–protein interactions Investigating the changes in the lipid organization produced by protein–lipid interactions, a GIXD study followed the structural monolayer changes produced by the binding of the B subunit of cholera toxin (CTB) [88]. Thus, GIXD investigation revealed a homogeneous lipid phase induced by the multivalent binding of the protein (CTB) to raft associated membrane receptors of mixed monolayers (DPPE:ganglioside GM1, 80:20). The authors established that the packing characteristics of this ‘textured’ lipid phase (LT) are intermediate of the well-known LO and gel lipid phases. The LT phase contained a rich variety of lipid tail tilt orientations from anisotropic and azimuthally swirled arrangements similar to the ones observed in macroscopic hexatic phases of liquid crystals. The XR data confirmed the binding of the protein to the monolayer (to the GM1 units) but without penetrating the lipid layer. The GIXD experiments on DPPE:GM1 supported bilayers revealed that these perturbations of the lipid order in the exterior membrane leaflet were communicated to the inner leaflet. The authors propose that such orientationally textured domains could have biological relevance for the lipid based signaling platforms and the cellular trafficking pathways. It could additionally offer an explanation of the mechanism of clathrin independent endocytosis of cholera toxin in which case the altered lipid packing is acting as the nucleation site for vesicle formation [88]. In line with the dual role played by cell membranes on inducing protein aggregation and membrane permeabilization, with strong implications for Alzheimer's disease, the mechanism of interaction of tau protein with two-dimensional model membrane systems was recently investigated [89]. The XR experiments indicated the presence of hTau40s protein (highly charged, soluble and yet highly surface active) under as well as in the DMPG monolayer, while GIXD data showed that the protein insertion disrupted the structural arrangement of the lipids. It was additionally observed that the disordered hTau40 protein partially adopted a more compact conformation by adsorbing at the bare air/water interface or to the DMPG monolayer. Additional neutron reflectivity experiments showed that the protein was able to completely disrupt supported DMPG bilayers, while no damage was found for the DPPC bilayer [89]. This strong interaction of hTau40s with anionic lipids, determining structural changes of the protein

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and membrane disruption, indicates two possible membrane-based mechanisms playing key roles in the tau aggregation and toxicity in neurodegenerative diseases. Another X-ray study on Langmuir monolayers highlighted the biotin-density dependence of the equilibrium structure and phase behavior of lipid-supported streptavidin assemblies which implies that the 2D crystallization of proteins can be induced as a densitydriven first-order phase transition [90]. 4.2.4. Lipid–NPs and lipid–polymer interactions The design and synthesis of new NPs for future biomedical applications has received increased scientific attention during the last years, yet the detailed understanding of their interaction with the cell membrane is still lacking. In this respect, the impact of Fe3O4 NPs, promising magnetic resonance imaging contrast enhancers and cell manipulation agents, on model membrane phospholipids has been investigated. Thus, the changes produced in DPPC Langmuir monolayers by Fe3O4 NPs (Fig. 8) capped with a biocompatible and stimuli-responsive ethylene glycol copolymer (MEO2MA90-co-OEGMA10) have been studied [91]. GIXD data revealed that the NPs (adsorbed from the subphase or co-spread with the lipid at the air/water interface) were able to change the condensed structure of the DPPC monolayers by increasing the inplane packing density (Fig. 8). Correlated to IRRAS data, this behavior was explained by the ability of the NPs to change the DPPC head group hydration and orientation. Moreover it is shown that dehydration effects, as well as the physical–chemical properties of the NPs are dictated by the copolymer [91]. Furthermore, the correlation of the GIXD results with Langmuir isotherms and TRXF data allowed to calculate the percentage of the interfacial area occupied by the NPs at different surface pressures in mixed DPPC–NP layers, including the percentage of squeezed out NPs. Indeed, as previously reported for pure NP layers [92,93], the NPs were trapped at the interface in the mixed DPPC–NP layer up to a critical surface pressure value, determined by the molar ratio of the two polymers. Above this critical surface pressure, the squeezing out of the NPs occurs [94] and the DPPC monolayer recovers its characteristic structural parameters above 35 mN/m, as proved by GIXD and IRRAS (Fig. 8) [91]. The finding that the NPs ability of insertion into model membranes is controlled by their surface activity could have

Fig. 8. A) Variation of the tilt angle t with the surface pressure π of a pure DPPC layer (water subphase – ▲; PBS, pH 7.4 – ■; Tris–HCl, pH 8 – ●) compared to that of a mixed DPPC – Fe3O4@ MEO2MA90-co-OEGMA10 NP layer ( ) obtained by co-spreading and to that of a mixed DPPC – MEO2MA90-co-OEGMA10 layer ( ) obtained by co-spreading on a water subphase at 20 °C. B) Variation of the TRXF intensity of the Fe Kα line (6.4 keV) with the surface pressure of the mixed DPPC–NPs layer. C) IRRA spectra of monolayers of pure DPPC (black line) and mixed DPPC–NPs (red line) at 20 mN/m and 45 mN/m. D) Schematic representation of the mixed DPPC–NP layers below the critical surface pressure and above the critical surface pressure. Figure adapted from [91].

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important practical implications since their surface activity can be tuned (by changing the molar ratio of the two polymers) and is stimuli dependent (temperature, ionic strength) [95]. Thus, since they are able to incorporate, to assist the recovery of the lipid packing and to desorb thereafter from the restored membrane, the study supports the possible application of these NPs as sealing agents of injured cell membranes. In a similar study, the interactions between amphiphilic block copolymers (monodisperse poly(ethylene oxide)-poly(butylene oxide) (EOBO)) [96] and DPPC have been explored at the air/water interface. In this case, the results showed that the EOBO copolymers interacted with DPPC through a mechanism similar to that of the poloxamer P188 [97]. Being incorporated into the lipid layer at low pressure, the copolymer is mainly phase separated but influences slightly the DPPC condensed phase structure, and is ejected from the interface into the subphase by compression as indicated by the isotherms. Moreover, the authors propose the replacement of poloxamers with EOBOs for different medical applications due to their enhanced surface activity which will allow lowering the dosages by orders of magnitude [96]. 4.2.5. Lipid–synaptic vesicle interactions Synaptic vesicles (SVs) are small, membrane-bound organelles that store neurotransmitters in presynaptic nerve endings. For understanding the changes of the membrane structure produced upon fusion with SVs and the role played in the process by Ca2 + ions and PIP2 (phosphatidylinositol-4,5-bisphosphate), a monolayer study was employed [98]. The structural changes of lipid monolayers (pure DPPC or DPPC/PIP mixtures) induced by the injection of SVs or Ca2 + ions into the subphase were monitored by XR and GIXD. The main findings consist of the observation of a significant Ca2+-dependent reorganization of the monolayer structure as well as corresponding changes in the electron density profile, in particular in the presence of PIP2 [98]. Extrapolating the results, the authors speculate that a revision of the current protein-centered view of neurotransmitter release is needed since the structural membrane changes induced by direct cation binding to lipids may regulate Ca2+-dependent synaptic vesicle fusion with the plasma membrane. 4.2.6. Lipid–drug and hormone interactions Hormones as regulatory biochemicals are produced by specific cells, glands, and tissues. They influence as chemical messengers many physiological and behavioral activities, such as digestion, metabolism, growth, reproduction, and so on. For a better understanding of hormone–membrane interaction mechanisms, the influence of plant hormones (phytohormones) on 2D model membranes has been studied. The negatively charged indole-3-acetic acid (IAA), belonging to the auxins, and the positively charged zeatin, belonging to the cytokinins, are plant-growth hormones. They have been used together with negatively charged (1,2-dimyristoyl-sn-glycero-3[phospho-L-serine], DMPS), positively charged (1,2-dipalmitoyl-3trimethylammoniumpropane, DPTAP) [99] and zwitterionic lipids (DPPC) [100]. Additionally, the effect of cadmium and selenium ions on the interactions between hormones and lipids was investigated. The results revealed that both IAA and zeatin led to an expansion of the lipid monolayer caused by the electrostatic interactions established between the hormones and the oppositely charged lipid layers, while the presence of ions led to competitive interactions of both solutes with oppositely charged lipid head groups [99]. Moreover, GIXD data clearly showed that the selenate ions induced monolayer condensation by neutralizing the positive net charge of mixed DPTAP/DPPC monolayers, whereas IAA molecules penetrated the lipid monolayer, causing its expansion/fluidization [100]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of drugs that provides analgesic and antipyretic (fever-reducing) effects, and, in higher doses, anti-inflammatory effects. The most prominent members are aspirin, ibuprofen and naproxen. The interaction of selected NSAIDs

with DPPC has been investigated in two-dimensional (monolayers at the air/water interface) and three-dimensional (multilayers in lipid/water dispersions) model systems [101]. GIXD, DSC, SAXS and WAXS measurements indicated that all used NSAIDs altered the temperature of the phase transition from gel to liquid-crystalline, the arylacetic acid derivatives (acemetacin, used for the treatment of osteoarthritis, rheumatoid arthritis, lower back pain, and relieving post-operative pain, and indomethacin, used as a prescription medication to reduce fever, pain, stiffness, and swelling) having the strongest effects. These findings have been explained by changes of the lipid head group hydration and were associated with the gastric injury induced by NSAIDs [101]. The interaction of many other pharmacologically active compounds has been investigated in two-dimensional model membrane phospholipids. Among them, Lupane type pentacyclic triterpenes (LTs – lupeol and betulinic acid) [102] which are natural products with a broad spectrum of therapeutic action (anticancer, antibiotic, and anti-inflammatory activity) proved to be miscible with DPPC and octadecyl-sphingomyelin (SM) in the whole range of mole ratios. In these two component mixtures, the LTs proved to induce disorder in the phospholipid monolayer, as revealed by the disappearance of the diffraction signals in the GIXD spectra [102]. 4.2.7. Lipid–β-cyclodextrin (β-CD) interaction Cyclodextrins are produced from starch by enzymatic conversion. They are used in food, pharmaceutical, drug delivery, and environmental engineering. A GIXD study investigated the interaction of β-CD (7-membered cyclic oligosaccharides) with different Langmuir one- or two-component monolayer systems (cholesterol (chol), 1-stearoyl-snglycero-3-phosphocholine (lyso-PC), 1,2-dipalmitpyl-sn-phosphocholine (DPPC), sphingomyelin (SM) and the SM/chol and DPPC/chol mixtures) [103]. The paper reveals the ability of β-CD to complex and to remove cholesterol and the one-chain phospholipid (lyso-PC) from onecomponent monolayers, yet it could not remove double-chain phospholipids (DPPC and SM). Moreover, no interaction has been observed between β-CD and DPPC, while the condensed monolayer structure of SM was modified (more distorted hexagonal lattice). For the two-component systems the β-CD showed a compositiondependent ability in withdrawing cholesterol. Thus, cholesterol could be partially removed from mixed SM/chol monolayers with a large excess of cholesterol (30:70) and the process stopped as the membrane composition approached the stable complex stoichiometry (SM/chol 2:1). Moreover the strong SM/chol interactions at 50:50 composition and the tight packing (at 30 mN/m) of the mixed monolayer impeded the cholesterol removal. β-CD was able to remove easier cholesterol from DPPC/chol mixtures as compared to the analogous SM/chol systems, yet the removal did not occur in mixed monolayers with excess of phospholipids (DPPC/chol 70/30) [103]. 4.3. Ions at Langmuir monolayers Langmuir monolayers proved to be extremely convenient systems for studying the interaction between charged head groups of amphiphilic molecules and ions present in the subphase. Thus, the phase transition and the appearance of an X-phase were revealed by GIXD in monolayers of docosanoic acid (also behenic acid, BA) spread on copper chloride salt solutions at two different pH values (5.5 and 7.5) [104]. The X-phase was characterized by untilted molecules arranged in a highly distorted hexagonal lattice with Bragg peak indexing opposite to the classical high-pressure S-phase of fatty acids. Moreover, such an X-phase was visualized for the first time by the BAM (Brewster angle microscopy) technique [104]. Another systematic GIXD study [105] was dedicated to the disordering effect of sodium salts of monovalent anions that span the lyotropic series (Hofmeister series) on DPPC monolayers at 12 °C. The results show that salts expand the monolayer, transform ordered

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phases into liquid-expanded ones and affect the ordering of the lipid chains and their tilt angle in the condensed phase. Surprisingly, large hydrophobic anions, such as PF6− (hexafluorophosphate) or TPB− (B(C6H5)− 4 , tetraphenylborate) forced the DPPC lipids to adopt an untilted conformation in the condensed phase (Fig. 9), an unprecedented finding for Langmuir monolayers of this phospholipid. The study proposes the existence of an additional ‘break’ in the ‘Hofmeister line’, which could be of particular importance for soft-matter systems [105]. The strong effect of electrolytes was proved by the study of a 2hydroxyacid (2-hydroxyoctadecanoic acid) in Langmuir monolayers. In this system, the additional OH group present at the α-position to the carboxylic acid functionality was designed to enhance the chelation process with applications in crystallization and biomineralization [106]. When compared to octadecanoic acid, an enhanced interfacial interaction was observed for 2-hydroxyoctadecanoic acid due to a complex balance of hydrogen bonding, electrostatics, and steric effects. All these interactions defined a complex phase behavior of the monolayers formed on different subphases. Thus, while on the pure water subphase hydrogen bonding dominated with three phases coexisting at low pressures, the introduction of calcium ions into the subphase ensured strong cation binding to the surfactant head groups through chelation. Very unstable monolayers were obtained in the presence of sodium bicarbonate subphases due to short-range hydrogen bonding interactions between the monolayer and bicarbonate ions facilitated by the sodium cation which enhanced the surfactant solubility [106]. Related to the neurotoxic effects produced by the accumulation of mercury in the central nervous system, a recent study focused on understanding the influence of mercury ions (HgCl2 and Hg(NO3)2) on model membrane phospholipids of DPPG, DPPC, 1-octadecyl-2-sn-phosphatidylcholine (lyso-PC), and SM [107]. Studying Langmuir monolayers by GIXD and XR it was revealed that the elastic properties of phospholipid monolayers are a key factor for the interaction with mercury ions. Thus, the mercury ion complexation had a stronger effect on the monolayer structure of phospholipids forming more compressible films (like SM and lyso-PC) as compared to condensed layers of low compressibility (such as DPPG and DPPC). This finding could have biological relevance since SM is one of the most abundant lipids found in neuron shells and therefore could be the target lipid for the toxic complexation with mercury ions [107]. 4.4. Chemical reactions in Langmuir monolayers For many years, hydrolysis reactions catalyzed by enzymes (phospholipases) have been studied at phospholipid monolayers. These enzymes are highly surface active and have a much higher affinity for substrate aggregates compared to monomeric substrates. The

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advantage of these studies was the possibility to change many physical parameters (packing density, phase state, ionization degree) of the lipid aggregate in an easy way. The yield of the hydrolysis reactions has been determined by IRRAS or PM-IRRAS as a function of these physical parameters and clear conclusions could be drawn about their influence on the activation and inhibition of the enzymes [108–112]. Another study was motivated by gaining a better understanding of the interaction of reactive oxygen radicals (ROS) with membrane models. ROS play an important role in different pathological pathways, as cancer development and even aging. DPPC monolayers have been studied during the attack of hydroxyl radicals (Fenton reaction) [113]. Based on the experimental data, the authors considered that the observed solidification of the monolayer was attained due to the preferential HO− radical attack to the DPPC head group. Thus, a partial cleavage of the head group (as indicated by IRRAS) led to a reduced head group size, a decreased intermolecular repulsion (lowering thus the phase transition pressure from the fluid to the condensed phase), an improved crystallinity of the condensed phase (as proved by GIXD) and the binding of Fe2+ to the monolayer suggesting an induced net negative lipid charge (as reflected by XR) [113]. Despite the fact that the concept of polymerization of Langmuir monolayers of amphiphiles containing diacetylene units was established a long time ago [114,115] it is only recently that the first functional carbon nanosheets with a uniform thickness have been obtained by this procedure. The carbon nanosheets were prepared by carbonization upon UV irradiation of Langmuir monolayers of amphiphiles containing hexayne segments (Fig. 10) [116]. The main achievement consists of the elegant carbonization of the monolayer at room temperature which yielded a graphitic carbon structure similar to that usually obtained upon annealing at extremely high temperatures (800 °C). The complex study reveals among other characterization details the importance of GIXD and IRRAS for defining the monolayer structure before and after carbonization. GIXD data (Fig. 10A–B) proved the coexistence of two structures (presumably from the uncarbonized and partially carbonized monolayer). These data corroborated with IRRA spectra (experimental) and their global fit (simulated spectra) allowing to develop a molecular picture of the uncarbonized monolayer structure. Details of the layer thickness and the different tilt angles of the hexayne (62.5°) and dodecyl (35°) segments in respect to the normal to the surface have been determined. Additionally, the IRRA spectra allowed to follow the carbonization reaction with the disappearance of the characteristic hexayne segment bands (2200 and 2172 cm−1 – Fig. 10D) and the transition upon carbonization of the dodecyl chains from a liquid-condensed state (methylene asymmetric and symmetric stretching vibrations at 2919 and 2849 cm−1, respectively) to a liquid-expanded one (methylene band positions at 2924 and 2855 cm−1) (Fig. 10C).

Fig. 9. Contour plots of the diffracted intensity versus the in-plane Qxy and out-of-plane Qz scattering vector components for a DPPC monolayer at 30 mN/m and 12 °C on: A) water, B) 5·10−6 M NaTPB, C) 5·10−5 M NaTPB, and D) 5·10−4 M NaTPB. Figure adapted from [105].

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Fig. 10. Chemical structure of the hexayne amphiphile investigated in Langmuir monolayers is shown above. A)–B) Contour plots of the corrected GIXD intensities as a function of the inplane (Qxy) and out-of-plane (Qz) scattering vector components showing two co-existing structures (supposedly from the uncarbonized (unc) and partially carbonized (pc) monolayer). The GIXD measurement was done on monolayers before UV irradiation. The carbonized monolayers displayed no GIXD signal. C)–D) IRRA spectra following the progress of monolayer carbonization recorded before (orange line) and after 1–40 min (gray and blue lines) of UV irradiation. C) Upon carbonization the methylene bands (2919 and 2849 cm−1) decreased in intensity and shifted to higher wavenumbers (2924 and 2855 cm−1, respectively). Thus, the carbonization led to a fluidization of the dodecyl chains as a result of the observed lateral expansion of the layer. D) The bands at 2200 and 2172 cm−1 associated with the hexayne segment vanished after 40 min of UV irradiation, suggesting an efficient carbonization of the monolayer. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4.5. Langmuir monolayers as templates 4.5.1. Biomineralization Inspired from nature, the biomineralization at or close to interfaces became in the last years a successful research field. Since cellular membranes have been considered potential templates for mineral nucleation (the so-called biomineralization process), Langmuir monolayers received more and more attention. Used as model systems, monolayers formed at the air/water interface proved very useful in revealing the mechanism, kinetics and key factors of the biomineralization process. Moreover, since it is considered that the accumulation of ions occurs at the charged membrane regions formed by proteins and lipids, the most encountered Langmuir template systems are those prepared from charged proteins or peptides, lipids or surfactants, polymers or their mixtures. Well-known examples of proteins involved in biomineralization are offered by collagen, a protein assembling into fibrils and fibers [117,118], and chitin, assembling into different phases and even nematic phases [119,120]. Organized protein structures at liquid interfaces can act as the starting point for nucleation and sometimes they become integrated into the newly formed composite material. The hydrophobins are another example of a class of amphiphilic proteins able to self-assemble at interfaces [121] and to serve as templates [122].

4.5.1.1. Biomineralization of calcium-phosphate and calcium carbonate. The fabrication of organic/inorganic hybrid surfaces in a controlled manner was successful for panoply of systems. Among the most studied biomineralization processes are those dedicated to calcium carbonate (often found as calcite and aragonite) [123] and to the structurally and chemically more complex calcium phosphate [124,125]. The concept of mineralization induced by a structural match between the interfaces of a template presenting arrayed functional groups and that of the nucleating crystal opened a new dimension to the use of Langmuir layers. Thus, in the context of biomaterials for applications in bone tissue regeneration, calcium-phosphate mineralization was monitored in Langmuir layers of three different amphiphilic and

acidic β-sheet-forming peptides [126] (Pro-Phe-(Asp-Phe)5-Pro (PFD5), Pro-Phe-(Glu-Phe)5-Pro (PFE-5) and Pro-Glu- (Phe-PSer)4-PheGlu-Pro (PPS)). It was known that the mineralization process in monolayers incubated on simulated body fluid (SBF) starts with the generation of calcium-phosphate nanometer-size clusters [127] that accumulate along with other ions at the templating surface and transform over time into the thermodynamically stable crystalline apatite form. In a study reported in [126] it is shown that according to the GIXD data, the peptide PFD-5 (decorated with PO2− 4 groups) monolayer interacts faster and to a larger extent with the mineralizing solution, losing the internal monolayer order during the mineralization process. In contrast, the more rigid PFE-5 and PPS peptide (decorated with COO− groups) films were able to conserve their structure during the mineralization process. These more rigid films formed thinner mineralized layers as amorphous calcium phosphate together with the apatite phase at PFE-5, and the apatite phase only at PPS monolayers. The study highlights the differences in mineralized film morphology and peptide lattice behavior of β-sheet templates induced by the nature of the negatively charged moiety: phosphate groups compared to carboxyl groups [126]. The nucleation and growth of calcium phosphate at the air/water interface were also achieved by using other Langmuir systems like surfactants [128], polymers [129] or mixed of lipid–polymer monolayers [130]. Moreover, key factors for the biomineralization of longrange and hierarchically organized calcium phosphate materials have been identified as: the monolayer charge, the nature of polyelectrolytes, the pH of the subphase (low pH adapted for the polycationic [131,132] monolayers and high pH for the polyanionic ones) as well as the flexibility of the monolayer. Indeed, the studies show that the more flexible polymer monolayers induced the formation of higher uniform calcium phosphate films as compared to the ones obtained under stiff surfactant monolayers. Similar to the calcium phosphate case, the biomineralization of CaCO3 at the air/water interface allowed the identification of the monolayer charge as a key player for the biomineralization process. Thus, while negatively charged monolayers of stearic acid induced the formation of homogeneous hybrid films, the uncharged layers of

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Fig. 11. A) High magnification SEM image of a flower-like calcium carbonate crystal grown under a pepsin Langmuir monolayer for 12 h. B) Schematic representation showing the control of the initial concentration of Ca2+ on the mineralization process and the final morphology of CaCO3 particles formed under monolayers of the amphiphilic polypeptide: poly(glutamic acid)22-block-poly(alanine)8. Figures adapted from [139,141].

stearyl alcohol induced the formation of only randomly distributed single crystals [133]. Moreover the biomineralization process of calcium carbonate was identified as occurring via pre-nucleation clusters [134,135]. Besides the monolayers of the above-mentioned surfactants, other systems like calixarenes and resorcarenes [136,137], as well as mixed surfactant monolayers (dioctadecyl dimethyl ammonium bromide and dodecyl sulfate sodium salt or heneicosanoic acid) [138] have been used as Langmuir templates for the biomineralization of CaCO3. Langmuir monolayers of pepsin (a proteolytic enzyme in the stomach responsible for the degradation of food proteins into peptides) have also been successfully used as biomimetic templates for the calcium carbonate mineralization from amorphous nanoparticles which grow into flower-like superstructures (Fig. 11A) [139]. Furthermore, the biomimetic mineralization of CaCO3 was proved to be possible even under a zwitterionic phospholipid (DPPC) monolayer. In this case, the transformation of the initial amorphous calcium carbonate nanoparticles into the metastable vaterite phase and finally into the thermodynamically stable calcite phase was observed [140]. Another study dedicated to the mineralization of CaCO3 at the air/water interface employed an amphiphilic polypeptide (poly(glutamic acid)22-block-poly(alanine)8 or PGlu22-b-PAla8) to play the double role of a soluble (functional) additive and insoluble (structural) matrix (Fig. 11B) [141]. In this case, the X-ray diffraction and Raman spectroscopy studies proved that the calcite polymorph was obtained. Moreover, the PGlu22-b-PAla8 peptide initiates the amorphous precursor phase and heterogeneous nucleation of CaCO3 at the air/water interface, while it temporarily stabilizes the gelatinous precursors as a process-directing agent. Yet, the second key factor of the mineralization process proved to be the initial concentration of Ca2+ which controls the crystallization and the final morphology of CaCO3 particles. 4.5.1.2. Biomineralization of iron-(hydr)oxide. An elaborate biomineralization study with Langmuir monolayers using X-ray methods (XR, TRXF, and GIXD) [142] aimed at understanding the growth mechanism of magnetite (Fe3O4) nano-crystals employed by magnetotactic bacteria. The work was dedicated to the accumulation of ferric iron Fe(III) or ferrous iron Fe(II) under dihexadecyl phosphate (DHDP) and arachidic acid (AA) monolayers. Interestingly, the X-ray reflectivity and fluorescence data of monolayers formed on FeCl3 aqueous subphases indicate remarkably high levels of surface-bound Fe3+ that exceed the amount necessary to neutralize theoretically completely deprotonated monolayers (DHDP or AA). Thus, it is suggested that nano-scale iron-(hydr)oxide complexes (oxides, hydroxides or

oxyhydroxides) are linking the head groups, overcompensating the maximum possible charges at the interface. The lack of in-plane ordering revealed by the GIXD experiments suggests the formation of an amorphous network of iron-(hydr)oxide linking neighboring head groups of amphiphiles. It is suggested that the binding of these complexes is done by chemical bonds rather than pure electrostatic/ thermodynamic forces and that the buffer used (Tris–KCl) favors the formation of larger nano-particles. For the pure FeCl2 solutions, a mixture of Fe(II) and Fe(III) characterized the system, suggesting the formation of magnetite (Fe3O4) and a lower amount of iron at the interface (Fig. 12) [142]. This ability of Fe3+ in forming iron complexes (Fe(OH)3 or Fe(OH)2+) and binding via covalent bonds even to uncharged monolayers is in contrast with the behavior of La3 + ions which are found to bind to negatively charged monolayers by purely electrostatic interactions [143]. In another paper [144], focused on ion specific effects of Fe3+ and La3 + at AA Langmuir monolayers, it is shown that the transition pressures from a tilted (L2) to an untilted (LS) phase is ion specific. Moreover, a much higher concentration of Fe3+ ions was found in the head group region compared to La3 + ions [144]. The same research group investigated the interfacial properties and iron binding to bacterial proteins which are able to promote the growth of magnetite nano-crystals. The monolayer study focused on the X-ray reflectivity and surface spectroscopy to determine the structural properties of the protein promoter of magnetite nano-crystal growth (Mms6 of Magnetospirillum magneticum AMB-1). The XR data revealed that the conformational changes of the protein at the interface are surface pressure and iron ion dependent processes. Moreover, TRXF allowed the quantitative determination of surface bound ions to the protein layer, highlighting that the ferric iron binds to Mms6 at higher densities compared to other ions such as Fe2+ or La3+ [145]. 4.5.2. Construction of functional nano-materials The ability of biological samples to self-assemble at the air/water interface makes them valuable templates for functional nano-materials. This provides a remarkably active research field at the interface between chemistry, biology, and materials science. Optically transparent films of interlinked gold nanofibers having electrical conductivity and pronounced surface enhanced Raman (SERS) activity were obtained upon incubation of positively charged β-sheet peptide LB monolayers in an aqueous solution of Au(SCN)−1 4 [146]. The successful gold deposition used a recently discovered chemical process involving spontaneous crystallization and reduction of

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Fig. 12. A) Reflectivity data for DHDP on various bulk solutions. B) Fluorescence intensity integrated below the critical angle over Qz = 0.01–0.021 Å−1 for DHDP on FeCl3–Tris–KCl (10−3 M FeCl3), FeCl2–Tris–KCl (10−3 M FeCl2) and pure Tris–KCl. C) Proposed representation of the iron binding. FeO6 octahedrons are linked together by corner sharing and form a gel-like structure. The oxygen atoms can be replaced by hydroxyl groups (OH) or water molecules. D) A single Fe(OH)2+ molecular binding to two phosphate groups. Figure adapted from [142].

water-soluble Au(SCN)−1 upon anchoring on the amine moieties 4 displayed on the surface. The procedure opened a promising avenue for the development of nano-structured films with practical applications [146]. In another study, Au nanoplates were successfully obtained under bovine serum albumin Langmuir monolayers [147]. Salbutamol sulfate crystals, commonly used as a bronchodilator for asthma and/or chronic obstructive pulmonary disease, were successfully grown beneath Langmuir monolayers of pure DPPC or a ‘mixed’ system (DPPC, POPG and PA) [148]. Powder X-ray diffraction measurements proved that the crystals exhibited a range of morphologies, dependent on the crystallization route, the nature of the template used (pure DPPC or mixture) and the monolayer lateral pressure. 5. Phospholipid bilayers at the solid–liquid interface For approaching even more the lipid bilayer architecture of cellular membranes, X-ray measurements have been developed for single phospholipid bilayers prepared at the solid/liquid (quartz crystal/water) interface [149]. In this case, GIXD and XR experiments revealed that the lateral ordering in a supported DPPE (1,2-dipalmitoyl-sn-glycero3-phosphoethanolamine) bilayer was weaker than that found in DPPE Langmuir monolayers. Moreover in contrast to the scattering from free standing PC bilayers, the bilayer leaflets proved to be uncoupled [149]. Later on, lipid bilayers on polyethylene glycol cushions have been developed and investigated by neutron and X-ray reflectivity measurements [150]. It was found that the hybrid bilayer/polyethylene glycol (PEG) systems formed a hydrated cushion beneath the bilayer only when the terminal ends of the lipopolymers were functionalized

with reactive end groups able to covalently bind (tether) to the underlying solid support. These reactive PEG tethered systems were able to cover the surface nearly completely with a bimodal distribution of heights of sub-micrometer lateral dimensions due to the existence of cushioned membrane domains and uncushioned regions. Yet, the cushioned membrane fraction could be tuned by adjusting the molar ratio of the lipopolymer in the bilayer system [150]. Moreover, the crystalline domain structure and the nucleation of cholesterol crystals has been recently studied in more complex single hydrated DPPC:Cholesterol:POPC bilayers prepared on polymer cushions of polyethyleneimine (PEI) [15]. GIXD measurements of these single hydrated bilayers have been compared to that of monolayers with the same composition. The results showed clear differences of the phases and structure of the crystalline domains. Thus, while in monolayers mixtures of DPPC and cholesterol formed single crystalline phases for all studied mixtures, the interactions established between the opposing leaflets of the bilayer system induced, depending on the lipid composition, phase separation, changes in molecular tilt angle, or formation of cholesterol crystals. In this case, monolayer thick ordered domains of cholesterol and DPPC were formed, each of the lipid leaflets diffracting independently, whereas for the mixtures with an excess of cholesterol (DPPC:Chol ratio b 46:54) cholesterol bilayer thick crystals could be identified. Interestingly, the nucleation of the cholesterol crystals occurred at concentrations close to the actual cell plasma membrane composition [15]. A following study highlighted the spontaneous formation of two- and three-dimensional cholesterol crystals in single hydrated lipid bilayers formed in another ternary system of ceramide/cholesterol/POPC at different concentrations

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[151]. An interesting structural comparison between monolayers, bilayers, and multilayers of lipids was recently reported in a minireview [152]. 6. Conclusions and outlook In this contribution, the relevance and utility of Langmuir monolayers at the soft air/liquid interface as suitable models to study physical and chemical interactions at membrane surfaces are demonstrated (see also [153,154]. Although Langmuir monolayers do not directly serve applications, they have gained increasing importance as in the last 2–3 decades a wealth of techniques to study such systems appeared with resolution from submolecular to supramolecular. Most important have been the optical microscopies, synchrotron based Xray methods and FTIR spectroscopy. There has been much progress in making use of those techniques, and this process will continue. On top one can also expect new information from the development of further methods like nonlinear optical and Raman techniques as well as from synthetic organic chemistry which provides sophisticated molecules able to closely mimic biological features. Even if the 2D monolayer represents only one half of a membrane, this model system is extremely useful to help in understanding the physics behind interactions at membrane surfaces. For many biological systems elucidation of structures and processes with submolecular resolution is necessary, and this can be afforded by means of Langmuir monolayers. On supramolecular scale there are many cooperative processes like signaling, energy conversion and locomotion awaiting to be understood and controlled. Along this line, different types of peptides and glycolipids allow studies of membrane processes like molecular recognition, cooperative binding and aggregation. As we have shown, the monolayer system is of outstanding value to study reactions at interfaces with the possibility to vary many parameters over a broad range. Therefore, although these systems are very synthetic they carry many promises not only to study general fluid interface physics, but also important membrane processes, which give them a bright future. The monolayer is of course not suited for the study of integral membrane proteins. Therefore, studies using bilayers and monolayers are not only complementary but also advantageous to answer important biophysical questions from different viewpoints. These answers are also increasingly demanded to achieve progress in medicine, biotechnology and many related sciences and their applications, and therefore we expect that studies with more refined model systems will encounter a bright future. Acknowledgments We are grateful to many colleagues for their inspiring collaboration at the beamline BW1 (HASYLAB, DESY, Hamburg, Germany) resulting in many high quality papers cited in this review. We thank HASYLAB for providing excellent conditions and support. This work was supported by the Max Planck Society. References [1] Singer SJ, Nicolson GL. The fluid mosaic model of the structure of cell membranes. Science 1972;175:720–31. [2] Kjaer K, Als-Nielsen J, Helm CA, Laxhuber LA, Möhwald H. Ordering in lipid monolayers studied by synchrotron X-ray diffraction and fluorescence microscopy. Phys Rev Lett 1987;58:2224–7. [3] Dutta P, Peng JB, Lin B, Ketterson JB, Prakash M, Georgopoulos P, et al. X-ray diffraction studies of organic monolayers on the surface of water. Phys Rev Lett 1987;58: 2228–31. [4] Penfold J, Thomas RK. The application of the specular reflection of neutrons to the study of surfaces and interfaces. J Phys Condens Matter 1990;2:1369–412. [5] Vaknin D, Kjaer K, Als-Nielsen J, Lösche M. Structural properties of phosphatidylcholine in a monolayer at the air/water interface. Neutron reflection study and reexamination of x-ray reflection measurements. Biophys J 1990;59:1325–32. [6] Reiter R, Motschmann H, Orendi H, Nemetz A, Knoll W. Ellipsometric microscopy. Imaging monomolecular surfactant layers at the air–water interface. Langmuir 1992;8:1784–8.

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Please cite this article as: Stefaniu C, et al, Langmuir monolayers as models to study processes at membrane surfaces, Adv Colloid Interface Sci (2014), http://dx.doi.org/10.1016/j.cis.2014.02.013

Langmuir monolayers as models to study processes at membrane surfaces.

The use of new sophisticated and highly surface sensitive techniques as synchrotron based X-ray scattering techniques and in-house infrared reflection...
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