Oxysterols in Cell Viability, Phospholipidosis and Extracellular Vesicles Production in a Lung Cancer Model.

The study carried out systematic research on the influence of selected oxysterols on cells viability, phospholipidosis and the level of secreted extracellular vesicles. Three oxidized cholesterol derivatives, namely 7α-hydroxycholesterol (7α-OH), 7- ketocholesterol (7-K) and 24(S)-hydroxycholesterol (24(S)-OH) were tested in three different concentrations: 50 µM, 100 µM and 200 µM for 24 h incubation with A549 lung cancer cell line. All the studied oxysterols were found to alter cells viability. The lowest survival rate of the cells was observed after 24 h of 7-K treatment, slightly better for 7α-OH while cells incubated with 24(S)-OH had the best survival rate among the oxysterols used. 7-K increased phospholipids accumulation in cells, however, most noticeable effect was noticed for 24(S)-OH. Changes in the level of extracellular vesicles secreted in cells culture after the treatment with oxysterols were also observed. It was found that all oxysterols used increased the level of secreted vesicles, both exosomes and ectosomes. The strongest effect was noticed for 24(S)-OH. Taken together, these results suggest that 7-K is the most potent inducer of cancer cell death, while 7α-OH is slightly less potent in this respect. The lower cytotoxic effect of 24(S)-OH correlates with greater phospholipids accumulation, extracellular vesicles production and better cells survival.

Lithocholic acid-based oligomers as drug delivery candidates targeting model of lipid raft.

This study presents a new approach to designing a lithocholic acid functionalized oligomer (OLithocholicAA-X) that can be used as a drug carrier with additional, beneficial activity. Namely, this novel oligomer can incorporate an anti-cancer drug due to the application of an effective backbone as its component (lithocholic acid) alone is known to have anticancer activity. The oligomer was synthesized and characterized in detail by nuclear magnetic resonance, attenuated total reflectance Fourier-transform infrared spectroscopy, ultraviolet-visible spectroscopy, thermal analysis, and mass spectrometry analysis. We selected lipid rafts as potential drug carrier-membrane binding sites. In this respect, we investigated the effects of OLithocholicAA-X on model lipid raft of normal and altered composition, containing an increased amount of cholesterol (Chol) or sphingomyelin (SM), using Langmuir monolayers and liposomes. The surface topography of the studied monolayers was additionally investigated by atomic force microscopy (AFM). The obtained results showed that the investigated oligomer has affinity for a system that mimics a normal lipid raft (SM:Chol 2:1). On the other hand, for systems with an excess of SM or Chol, thermodynamically unfavorable fluidization of the films occurs. Moreover, AFM topographies showed that the amount of SM determines the bioavailability of the oligomer, causing fragmentation of its lattice.

Insight into the Molecular Mechanism of Surface Interactions of Phosphatidylcholines─Langmuir Monolayer Study Complemented with Molecular Dynamics Simulations.

Mutual interactions between components of biological membranes are pivotal for maintaining their proper biophysical properties, such as stability, fluidity, or permeability. The main building blocks of biomembranes are lipids, among which the most important are phospholipids (mainly phosphatidylcholines (PCs)) and sterols (mainly cholesterol). Although there is a plethora of reports on interactions between PCs, as well as between PCs and cholesterol, their molecular mechanism has not yet been fully explained. Therefore, to resolve this issue, we carried out systematic investigations based on the classical Langmuir monolayer technique complemented with molecular dynamics simulations. The studies involved systems containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) analogues possessing in the structure one or two polar functional groups similar to those of DPPC. The interactions and rheological properties of binary mixtures of DPPC analogues with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol were compared with reference systems (DPPC/POPC and DPPC/cholesterol). This pointed to the importance of the ternary amine group in PC/cholesterol interactions, while in PC mixtures, the phosphate group played a key role. In both cases, the esterified glycerol group had an effect on the magnitude of interactions. The obtained results are crucial for establishing structure-property relationships as well as for designing substitutes for natural lipids.

Advantages of the classical thermodynamic analysis of single-and multi-component Langmuir monolayers from molecules of biomedical importance-theory and applications.

The Langmuir monolayer technique has been successfully used for decades to model biological membranes and processes occurring at their interfaces. Classically, this method involves surface pressure measurements to study interactions within membrane components as well as between external bioactive molecules (e.g. drugs) and the membrane. In recent years, surface-sensitive techniques were developed to investigate monolayers in situ; however, the obtained results are in many cases insufficient for a full characterization of biomolecule-membrane interactions. As result, description of systems using parameters such as mixing or excess thermodynamic functions is still relevant, valuable and irreplaceable in biophysical research. This review article summarizes the theory of thermodynamics of single- and multi-component Langmuir monolayers. In addition, recent applications of this approach to characterize surface behaviour and interactions (e.g. orientation of bipolar molecules, drug-membrane affinity, lateral membrane heterogeneity) are presented.

Site of the Hydroxyl Group Determines the Surface Behavior of Bipolar Chain-Oxidized Cholesterol Derivatives─Langmuir Monolayer Studies Supplemented with Theoretical Calculations.

Cholesterol oxidation products (called oxysterols) are involved in many biological processes, showing both negative (e.g., neurodegenerative) and positive (e.g., antiviral and antimicrobial) effects. The physiological activity of oxysterols is undoubtedly closely related to their structure (i.e., the type and location of the additional polar group in the cholesterol skeleton). In this paper, we focus on determining how a seemingly minor structural change (introduction of a hydroxyl moiety at C(24), C(25), or C(27) in the isooctyl chain of cholesterol) affects the organization of the resulting molecules at the phase boundary. In our research, we supplemented the classic Langmuir monolayer technique, based on the surface pressure and electric surface potential isotherms, with microscopic (BAM) and spectroscopic (PM-IRRAS) techniques, as well as theoretical calculations (DFT and MD). This allowed us to show that 24-OH behaves more like cholesterol and forms stable, rigid monolayers. On the other hand, 27-OH, similar to 25-OH, undergoes the phase transition from monolayer to bilayer structures. Theoretical calculations enabled us to conclude that the formation of bilayers from 27-OH or 25-OH is possible due to the hydrogen bonding between adjacent oxysterol molecules. This observation may help to understand the factors responsible for the unique biological activity (including antiviral and antimicrobial) of 27-OH and 25-OH compared to other oxysterols.

Comprehensive Approach to the Interpretation of the Electrical Properties of Film-Forming Molecules.

This paper presents a general protocol for the interpretation of the electric surface potential of Langmuir monolayers based on a three-layer capacitor model. The measured values were correlated with the results from DFT molecular dynamics simulations, and, as a result, the local dielectric permittivities and dipole-moment components of molecules organized in the monolayer were obtained. The main advantage of the developed approach is applicability to amphiphiles of any type; irrespective of the structure of the polar head as well as the molecular organization and inclination in the surface film. The developed methodology was successively applied to an atypical surface-active compound, perfluorodecyldecane, and its derivatives containing the hydroxyl, thiol, and carboxyl moiety. The following contributions to the apparent dipole moments connected with the reorientation of water molecules and local dielectric permittivities in the vicinity of polar and apolar molecule parts, respectively, were determined: µw/εw = -0.85 D, εp = 5.00, and εa = 1.80. Moreover, the investigated perfluorodecyldecane derivatives were comprehensively characterized in terms of their surface activity, film rheology, and effective surface dissociation equilibria. The proposed methodology may be crucial for the process of the design and the preliminary characterization of molecules for sensor and material science applications.

Oxysterols can act antiviral through modification of lipid membrane properties - The Langmuir monolayer study.

In this paper we tested how oxysterols influence on fusion process between viral lipid envelope and host cells membranes. For this purpose, the Zika virus was selected, while dendritic cell (DC) and neural cell (NC) membranes were chosen as target membranes. The investigated systems were modeled as multicomponent Langmuir monolayers and characterized using surface manometry and imaging in micro- (Brewster angle microscopy, BAM) and nanoscale (Atomic Force Microscopy, AFM) to monitor local heterogeneity. The fusion process was conducted by mixing viral and host cell membranes devoid and in the presence of oxysterols: 25-hydroxycholesterol (25-OH) and 7ß-hydroxycholesterol (7ß-OH) as representatives of chain- and ring-oxidized oxysterols, respectively. Our results show that oxysterols hinder the fusion with host cell membranes by modifying their biophysical properties. Moreover, oxysterols applied to an already infected membrane reverse the changes caused by the infection. It could therefore be concluded that oxysterols may display antiviral activity in two ways: they prevent the healthy membrane from viral infection by blocking the fusion process; and protect already infected membrane from pathological changes induced by the virus.

Different effects of oxysterols on a model lipid raft - Langmuir monolayer study complemented with theoretical calculations.

Three oxysterols (7ß-hydroxycholesterol; 7ß-OH, 7-ketocholesterol; 7-K and 25-hydroxycholesterol, 25-OH) differing in the site of oxidation (ring system versus chain) and kind of polar group (hydroxyl versus carbonyl) were studied in lipid raft environment using the Langmuir monolayer technique complemented with theoretical calculations. Experiments were performed for the unmodified raft system, composed of sphingomyelin (SM) and cholesterol (Chol), and in the next step the raft was modified by the incorporation of oxysterol in different proportions. In the examined three-component system (Chol:SM:oxysterol), apart from interactions between the lipid raft components, the affinity of Chol to its oxidized derivatives also plays an important role. 25-OH was found to enhance interactions between SM and Chol and thus stabilize the raft, contrary to 7ß-OH and 7-K, which exerted the fluidizing effect as well as the destabilization of the raft. Different action of oxysterols on model raft was observed. 7ß-OH and 7-K, which are highly potent inducers of cell death caused raft destabilization, while 25-OH, which is the least toxic of the investigated oxysterols, was found to stabilize the raft.

Predicting the packing parameter for lipids in monolayers with the use of molecular dynamics.

Lipid molecules form the backbone of biological membranes. Due to their amphiphilic structure, they can self-organize in a plethora of different structures when in contact with water. The type of self-assembled structure and its curvature depend on so-called shape factor or critical packing parameter, CPP, that can be derived knowing the molecular volume of a lipid (V), optimal surface area (a0) and critical chain length (lc) (see Intermolecular and Surface Forces by Jacob N. Israelachvili, Third Edition, 2011). The value of CPP allows not only to predict the type of self-assembled structure but also is a key factor for molecular interactions, which play a great role both in physiological and pathological conditions. The greatest difficulties arise when calculating the a0 parameter, and although for some typical membrane lipids these values have been determined, there are a number of derivatives for which this parameter, and thus CPP, are unknown. The value of CPP allows not only to predict the type of self-assembled structure but also is a key factor for molecular interactions, which play a great role both in physiological and pathological conditions. So far, the determination of the packing parameter required the use of theoretical models with assumptions deviating from the physical conditions. Here we report a method based on molecular dynamics, which was applied to simulate lipid membranes consisting of cholesterol, oxysterols, sphingolipids, phosphatidylcholines, and phosphatidylethanolamines. For lipid molecules for which CPPs have already been determined, high compliance has been demonstrated. This proves that the method presented herein can be successfully used to determine packing parameters for other membrane lipids and amphiphilic molecules.

Can oxysterols work in anti-glioblastoma therapy? Model studies complemented with biological experiments.

Despite the progress made in recent years in the field of oncology, the results of glioblastoma treatment remain unsatisfactory. In this paper, cholesterol derivatives - oxysterols - have been investigated in the context of their anti-cancer activity. First, the influence of three oxysterols (7-K, 7ß-OH and 25-OH), differing in their chemical structure, on the properties of a model membrane imitating glioblastoma multiforme (GBM) cells was investigated. For this purpose, the Langmuir monolayer technique was applied. The obtained results clearly show that oxysterols modify the structure of the membrane by its stiffening, with the 7-K effect being the most pronounced. Next, the influence of 7-K on the nanomechanical properties of glioblastoma cells (U-251 line) was verified with AFM. It has been shown that 7-K has a dose-dependent cytotoxic effect on glioblastoma cells leading to the induction of apoptosis as confirmed by viability tests. Interestingly, significant changes in membrane structure, characteristic for phospholipidosis, has also been observed. Based on our results we believe that oxysterol-induced apoptosis and phospholipidosis are related and may share common signaling pathways. Dysregulation of lipids in phospholipidosis inhibit cell proliferation and may play key roles in the induction of apoptosis by oxysterols. Moreover, anticancer activity of these compounds may be related to the immobilization of cancer cells as a result of stiffening effect caused by oxysterols. Therefore, we believe that oxysterols are good candidates as new therapeutic molecules as an alternative to the aggressive treatment of GBM currently in use.

From glioblastoma to COVID-19 ­ role of oxysterols in the human organism / Od glejaka po COVID-19 - rola oksysteroli w organizmie czlowieka.

Cholesterol is a precursor molecule for vitamin D, bile acids, and steroid hormones. Its oxidized forms, called oxysterols are by-products for synthesis, but also regulate cholesterolâs metabolism through different cell receptors. Cholesterol and oxysterols are important cell membrane components. Oxysterols show more biological activity than cholesterol itself, due to their pleiotropic cell effects. Oxysterol, contrary to cholesterol can cross the blood-brain barier and influence the nervous system. The etiology and pathogenesis of cancer can also be influenced by oxysterols. Elevated levels of oxysterol is often observed in many pathological state: diabetes, atherosclerosis, dementia, or endometriosis. Cholesterolâs oxidized forms can regulate immunity by activation or suppression of the immune cell during virus infection and inflammation.

25-hydroxycholesterol interacts differently with lipids of the inner and outer membrane leaflet - The Langmuir monolayer study complemented with theoretical calculations.

25-hydroxycholesterol (25-OH), a molecule with unusual behavior at the air/water interface, being anchored to the water surface alternatively with a hydroxyl group at C(3) or C(25), has been investigated in mixtures with main membrane phospholipids (phosphatidylcholines - PCs, and phosphatidylethanolamines - PEs), characteristic of the outer and inner membrane leaflet, respectively. To achieve this goal, the classical Langmuir monolayer approach based on thermodynamic analysis of interactions was conducted in addition to microscopic imaging of films (in situ with BAM and after transfer onto mica with AFM), surface-sensitive spectroscopy (PM-IRRAS), as well as theoretical calculations. Our results show that the strength of interactions is primarily determined by the kind of polar group (strong, attractive interactions leading to surface complexes formation were found to occur with PCs while weak or repulsive ones with PEs). Subsequently, the saturation of phosphatidylcholines apolar chain(s) was found to be crucial for the structure of the formed complexes. Namely, saturated PC (DPPC) does not have preferences regarding the orientation of 25-OH molecule in surface complexes (which results in the two possible 25-OH arrangements), while unsaturated PC (DOPC) enforces one specific orientation of oxysterol (with C(3)-OH group). Our findings suggest that the transport of 25-OH between inner and outer membrane leaflet can proceed without orientation changes, which is thermodynamically advantageous. This explains results found in real systems showing significant differences in the rate of transmembrane transport of 25-OH and the other chain-oxidized oxysterols compared to their ring-oxidized analogues or cholesterol.

How the replacement of cholesterol by 25-hydroxycholesterol affects the interactions with sphingolipids: The Langmuir Monolayer Study complemented with theoretical calculations.

In this paper, a representative of chain-oxidized sterols, 25-hydroxycholesterol (25-OH), has been studied in Langmuir monolayers mixed with the sphingolipids sphingomyelin (SM) and ganglioside (GM1) to build lipid rafts. A classical Langmuir monolayer approach based on thermodynamic analysis of interactions was complemented with microscopic visualization of films (Brewster angle microscopy), surface-sensitive spectroscopy (polarization modulation-infrared reflection-absorption spectroscopy) and theoretical calculations (density functional theory modelling and molecular dynamics simulations). Strong interactions between 25-OH and both investigated sphingolipids enabled the formation of surface complexes. As known from previous studies, 25-OH in pure monolayers can be anchored to the water surface with a hydroxyl group at either C(3) or C(25). In this study, we investigated how the presence of additional strong interactions with sphingolipids modifies the surface arrangement of 25-OH. Results have shown that, in the 25-OH/GM1 system, there are no preferences regarding the orientation of the 25-OH molecule in surface complexes and two types of complexes are formed. On the other hand, SM enforces one specific orientation of 25-OH: being anchored with the C(3)-OH group to the water. The strength of interactions between the studied sphingolipids and 25-OH versus cholesterol is similar, which indicates that cholesterol may well be replaced by oxysterol in the lipid raft system. In this way, the composition of lipid rafts can be modified, changing their rheological properties and, as a consequence, influencing their proper functioning.

Electrical Properties of Membrane Phospholipids in Langmuir Monolayers.

Experimental surface pressure (π) and electric surface potential (ΔV) isotherms were measured for membrane lipids, including the following phosphatidylcholines (PCs)-1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC); and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In addition, other phospholipids, such as phosphatidylethanolamines (represented by 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)) and sphingolipids (represented by N-(hexadecanoyl)-sphing-4-enine-1-phosphocholine (SM)) were also studied. The experimental apparent dipole moments (µAexp) of the abovementioned lipids were determined using the Helmholtz equation. The particular contributions to the apparent dipole moments of the investigated molecules connected with their polar (µ⟂p) and apolar parts (µ⟂a) were theoretically calculated for geometrically optimized systems. Using a three-layer capacitor model, introducing the group's apparent dipole moments (calculated herein) and adopting values from other papers to account for the reorientation of water molecules (µ⟂w/εw), as well as the for the local dielectric permittivity in the vicinity of the polar (εp) and apolar (εa) groups, the apparent dipole moments of the investigated molecules were calculated (µAcalc). Since the comparison of the two values (experimental and calculated) resulted in large discrepancies, we developed a new methodology that correlates the results from density functional theory (DFT) molecular modeling with experimentally determined values using multiple linear regression. From the fitted model, the following contributions to the apparent dipole moments were determined: µ⟂w/εw=-1.8±1.4 D; εp=10.2±7.0 and εa=0.95±0.52). Local dielectric permittivity in the vicinity of apolar groups (εa) is much lower compared to that in the vicinity of polar moieties (εp), which is in line with the tendency observed by other authors studying simple molecules with small polar groups. A much higher value for the contributions from the reorientation of water molecules (µ⟂w/εw) has been interpreted as resulting from bulky and strongly hydrated polar groups of phospholipids.

Molecular insight into neurodegeneration - Langmuir monolayer study on the influence of oxysterols on model myelin sheath.

Systematic studies on the influence of selected ring-oxidized (7α-hydroxycholesterol, 7α-OH; 7ß-hydroxycholesterol, 7ß-OH; 7-ketocholesterol, 7-K) and chain-oxidized (25-OH) sterols on lipid layer of myelin were performed. Myelin sheath was modeled as five-component Langmuir monolayer (Chol:PE:SM:PS:PC 50:20:12:9:9). Particular oxysterols have been incorporated into the model myelin sheath by replacing cholesterol totally or partially (1:1). The effect of oxysterol incorporation was characterized with surface pressure and electric surface potential - area isotherms and visualized with Brewster angle microscopy (BAM) and atomic force microscopy (AFM). It has been noticed that model myelin loses its homogeneous structure (due to the appearance of domains) at physiological bilayer conditions (30-35 mN/m). In the presence of oxysterols, the fluidity of myelin model increases and the organization of lipids is altered, which is reflected in the decrease of electric surface potential changes (ΔV). The strongest myelin/oxysterol interactions have been observed for 7-K and 25-OH, being the most cytotoxic oxysterols found in biological tests.

Unusual Behavior of the Bipolar Molecule 25-Hydroxycholesterol at the Air/Water Interface-Langmuir Monolayer Approach Complemented with Theoretical Calculations.

In this study, 25-hydroxycholesterol (25-OH), a biamphiphilic compound with a wide range of biological activities, has been investigated at the air/water interface. We were interested in how two hydroxyl groups attached at distal positions of the 25-OH molecule (namely, at C(3) in the sterane system and at C(25) in the side chain) influence its surface behavior. Apart from traditional Langmuir monolayers, other complementary surface-sensitive techniques, such as electric surface potential measurements, Brewster angle microscopy (BAM, enabling texture visualization and film thickness measurements), and polarization modulation-infrared reflection-absorption spectroscopy (PM-IRRAS), were applied. Experimental data have been interpreted with the aid of theoretical study. Our results show that 25-OH molecules in the monomolecular layer are anchored to the water surface alternatively with C(3) or C(25) hydroxyl groups. Theoretical calculations revealed that the populations of these alternative orientations were not equal and molecules anchored with C(3) hydroxyl groups were found to be in excess. As a consequence of such an arrangement, surface films of 25-OH are of lower stability as compared to cholesterol (considered as a non-oxidized analogue of 25-OH). Moreover, it was found that, upon compression, the transition from mono- to bilayer occurred. The molecular mechanism and interactions stabilizing bilayer structure were proposed. The explanation of the observed unusual surface behavior of 25-OH may contribute to an understanding of differences in biological activity between chain- and ring-oxidized sterols.

Effect of selected B-ring-substituted oxysterols on artificial model erythrocyte membrane and isolated red blood cells.

In this paper, systematic studies concerning the influence of selected oxysterols on the structure and fluidity of human erythrocyte membrane modeled as Langmuir monolayers have been performed. Three oxidized cholesterol derivatives, namely 7α-hydroxycholesterol (7α-OH) 7ß-hydroxycholesterol (7ß-OH) and 7-ketocholesterol (7-K) have been incorporated in two different proportions (10 and 50%) into artificial erythrocyte membrane, modeled as two-component (cholesterol:POPC) Langmuir monolayer. All the studied oxysterols were found to alter membrane fluidity and the effect was more pronounced for higher oxysterol content. 7α-OH increased membrane fluidity while opposite effect was observed for 7ß-OH and 7-K. Experiments performed on model systems have been verified in biological studies on red blood cells (RBC). Consistent results have been found, i.e. under the influence of 7α-OH, the elasticity of erythrocytes increased, and in the presence of other investigated oxysterols - decreased. The strongest effect was noticed for 7-K. Change of membrane elasticity was associated with the change of erythrocytes shape, being most noticeable under the influence of 7-K.

Perfluorohexyloctane (F6H8) as a delivery agent for cyclosporine A in dry eye syndrome therapy - Langmuir monolayer study complemented with infrared nanospectroscopy.

One of the key challenges in dry eye syndrome therapy is to find a suitable carrier for immunosuppressant drug - cyclosporine A (CsA) - delivery to the eye. To investigate this issue, herein we present a methodology based on the combined analysis in macro- (Langmuir monolayers), micro- (Brewster angle microscopy) and nanoscale (atomic force microscopy and infrared nano-spectroscopy). The applied approach proves that CsA affects the phospholipid part of the tear film lipid layer by loosening molecular packing. This effect can be reversed by the addition of perfluorohexyloctane (F6H8). We have highlighted that F6H8 increases the availability of CsA and therefore is appropriate carrier for CsA topical delivery to the eye in the dry eye syndrome. In addition, the applied herein procedure provides a simple, low-cost laboratory tool for preliminary studies involving membrane active pharmaceuticals, preceding in vivo tests.

Surface interactions determined by stereostructure on the example of 7-hydroxycholesterol epimers - The Langmuir monolayer study.

Stereoselective interactions are pivotal for molecular recognition between biomolecules and lipid surfaces. The aim of this study was to determine factors differencing molecular interactions between 7-hydroxycholesterol epimers (oxysterols, which excessively appear in pathological processes in human body) and natural membrane phospholipids. Two-component systems of different mutual proportions of 7-hydroxycholesterol (7α-hydroxycholesterol or 7-ß-hydroxycholesterol, in short 7α-OH or 7ß-OH) and membrane lipids (POPC, DPPC, DPPE, DPPS, SM) were systematically analyzed in artificial membranes modeled as Langmuir monolayers. Classical surface pressure measurements were complemented with direct visualization of films texture both in situ (with Brewster angle microscopy, BAM) and after their transfer onto solid supports (with Atomic Force Microscopy, AFM). Our results clearly show striking differences in surface properties of the studied binary mixtures, emphasizing distinct effects of both 7-hydroxycholesterol epimers on the organization of lipid layers. Systematic study allowed to conclude that the structure of polar head group and interfacial region of the molecule play important role in oxysterol-phospholipid interactions, while the hydrophobic region is significantly less important in this respect.

High-resolution label-free studies of molecular distribution and orientation in ultrathin, multicomponent model membranes with infrared nano-spectroscopy AFM-IR.

Biological membranes are undoubtedly very interesting systems. Unfortunately, their analysis is very complicated and therefore simplified artificial models, like Langmuir monolayers, are frequently applied. In this work a novel method of label-free monomolecular films analysis based on infrared nano-spectroscopy (AFM-IR) is presented. In order to verify applicability of this approach well-defined referential system of sphingomyelin (SM), cholesterol (Chol) and cyclosporin A (CsA) is applied. AFM-IR method allows to directly and chemoselectively map the distribution of components in model lipid membranes. Additionally the orientation of Chol and SM molecules in the monolayer is determined by application of two perpendicular infrared laser polarizations. This paper is the first report of using AFM-IR to analyze LB films with extremely high sensitivity.