Statin Action Targets Lipid Rafts of Cell Membranes: GIXD/PM-IRRAS Investigation of Langmuir Monolayers.

Lipid rafts are condensed regions of cell membranes rich in cholesterol and sphingomyelin, which constitute the target for anticholesterolemic drugs - statins. In this work, we use for the first time a combined grazing-incidence X-ray diffraction (GIXD)/polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS)/Brewster angle microscopy (BAM) approach to show the statin effect on model lipid rafts and its components assembled in Langmuir monolayers at the air-water interface. Two representatives of these drugs, fluvastatin (FLU) and cerivastatin (CER), of different hydrophobicity were chosen, while cholesterol (Chol) and sphingomyelin (SM), and their 1:1 mixture were selected to form condensed monolayers of lipid rafts. The effect of statins on the single components of lipid rafts indicated that both the hydrophobicity of the drugs and the organization of the layer determined the drug-lipid interaction. For cholesterol monolayers, only the most hydrophobic CER was effectively changing the film structure, while for the less organized sphingomyelin, the biggest effect was observed for FLU. This drug affected both the polar headgroup region as shown by PM-IRRAS results and the 2D crystalline structure of the SM monolayer as evidenced by GIXD. Measurements performed for Chol/SM 1:1 models proved also that the statin effect depends on the presence of Chol-SM complexes. In this case, the less hydrophobic FLU was not able to penetrate the binary layer at all, while exposure to the hydrophobic CER resulted in the phase separation and formation of ordered assemblies. The changes in the membrane properties were visualized by BAM images and GIXD patterns and confirmed by thermodynamic parameters of hysteresis in the Langmuir monolayer compression-decompression experiments.

Interfacial Behavior of Cubosomes: Combined Langmuir-Blodgett/Langmuir-Schaefer and AFM Investigations.

The Langmuir technique was applied for the first time to compare the layers obtained by spreading lipid liquid-crystalline nanoparticles monoolein 1-oleoyl-rac-glycerol (GMO)/Pluronic F108 cubosomes with the monolayers obtained by mixing the same components in chloroform at the air-water interface. The differences in the monolayer behavior and in the acting intermolecular forces were examined. The similarity of the isotherms obtained for the mixed components system and the cubosome-derived layer proved the disintegration of cubosomes into a single monolayer upon contact with the air-water interface. Despite the low Pluronic F108 content in both types of layers, a strong structural role of this stabilizer was also demonstrated. Cubosome-derived systems supported on hydrophilic mica substrates were prepared either using the combined Langmuir-Blodgett and Langmuir-Schaefer technique or via direct adsorption from the solution. The topographies of the obtained layers were studied by atomic force microscopy (AFM). Images obtained in the air mode revealed the disintegration of cubosomes and the formation of large crystallized structures of the polymer, while AFM imaging performed in water confirmed the presence of intact cubosomes on the surface of mica. We proved that the original structure of cubosomes remains on one condition: the films must not dry out; therefore, the aqueous environment must be preserved. This new approach provides an explanation in the ongoing discussion of what happens to lipid nanoparticles with or without cargo when they come into contact with an interface.

Revealing the structure and mechanisms of action of a synthetic opioid with model biological membranes at the air-water interface.

Synthetic opioids such as piperazine derivative called MT-45 interact with opioid receptors in a manner similar to morphine leading to euphoria, a sense of relaxation and pain relief and are commonly used as substituents of natural opioids. In this study we show the changes in the surface properties of nasal mucosa and intestinal epithelial model cell membranes formed at the air - water interface using Langmuir technique upon the exposure to MT-45. Both membranes constitute the first barrier to absorb this substance into the human body. The presence of the piperazine derivative affects the organization of both DPPC and ternary DMPC:DMPE:DMPS monolayers treated as simple models of nasal mucosa and intestinal cell membranes, respectively. This novel psychoactive substance (NPS) leads to the fluidization of the model layers, which may indicate their increased permeability. MT-45 has a greater influence on the ternary monolayers characteristic of the intestinal epithelial cells than nasal mucosa. It might be attributed to the increased attractive interactions between the components of the ternary layer, which in turn increase the interactions with a synthetic opioid. Additionally, the crystal structures of MT-45 determined by single-crystal and powder X-ray diffraction methods allowed us to both provide useful data for facilitating the identification of synthetic opioids as well as to attribute the effect of MT-45 to the ionic interactions between protonated nitrogen atoms and negatively charged parts of the polar heads of the lipids.

Model Lipid Raft Membranes for Embedding Integral Membrane Proteins: Reconstitution of HMG-CoA Reductase and Its Inhibition by Statins.

For the first time, HMG-CoA reductase, the membrane protein responsible for cholesterol synthesis, was incorporated into a lipid membrane consisting of DOPC:Chol:SM at a 1:1:1 molar ratio, which mimics the lipid rafts of cell membranes. The membrane containing the protein was generated in the form of either a proteoliposomes or a film obtained by spreading the proteoliposomes at the air-water interface to prepare a protein-rich and stable lipid layer over time. The lipid vesicle parameters were characterized using dynamic light scattering (DLS) and fluorescence microscopy. The incorporation of HMG-CoA reductase was reflected in the increased size of the proteoliposomes compared to that of the empty liposomes of model rafts. Enzyme reconstitution was confirmed by measuring the activity of NADPH, which participates in the catalytic process. The thin lipid raft films formed by spreading liposomes and proteoliposomes at the air-water interface were investigated using the Langmuir technique. The activities of the HMG-CoA reductase films were preserved over time, and the two lipid raft systems, nanoparticles and films, were exposed to solutions of fluvastatin, a HMG-CoA reductase inhibitor commonly used in the treatment of hypercholesterolemia. Both lipid raft systems constructed were useful membrane models for the determination of reductase activity and for monitoring the statin inhibitory effects and may be used for investigating other integral membrane proteins during exposure to inhibitors/activators considered to be potential drugs.

Influence of lipophilicity of anthracyclines on the interactions with cholesterol in the model cell membranes - Langmuir monolayer and SEIRAS studies.

The interactions of anthracyclines with biological membranes strongly depend on the drug lipophilicity, which might also determine the specific affinity to cholesterol molecules. Therefore, in this work we show the studies concerning the effect of two selected anthracyclines, daunorubicin (DNR) and idarubicin (IDA) on simple models of healthy (DMPC:Chol 7:3) and cancer cells membranes with increased level of cholesterol (DMPC:Chol 3:7) as well as pure cholesterol monolayers prepared at the air-water interface and supported on gold surface. It has been shown that more lipophilic IDA is able to penetrate cholesterol monolayers more effectively than DNR due to the formation of IDA-cholesterol arrangements at the interface, as proved by the thermodynamic analysis of compression-expansion cycles. The increased interactions of IDA were also confirmed by the time measurements of pre-compressed monolayers exposed to drug solutions as well as grazing incidence X-ray diffraction studies demonstrating differences in the 2D organization of cholesterol monolayers. Langmuir studies of mixed DMPC:Chol membranes revealed the reorganization of molecules in the cancer cell models at the air-water interface at higher surface pressures due to the removal of DNR, while increased affinity of IDA towards cholesterol allowed this drug to penetrate the layer more efficiently without its removal. The SEIRAS spectra obtained for supported DMPC:Chol bilayers proved that IDA locates both in the ester group and in the acyl chain region of the bilayer, while DNR does not penetrate the membranes as deeply as IDA. The increased penetration of the mixed phospholipid layers by idarubicin might be attributed to the higher lipophilicity caused by the lack of methoxy group and resulting in a specific affinity towards cholesterol.

Designing a Useful Lipid Raft Model Membrane for Electrochemical and Surface Analytical Studies.

A model biomimetic system for the study of protein reconstitution or drug interactions should include lipid rafts in the mixed lipid monolayer, since they are usually the domains embedding membrane proteins and peptides. Four model lipid films composed of three components: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (Chol) and sphingomyelin (SM) mixed in different molar ratios were proposed and investigated using surface pressure measurements and thermodynamic analysis of the monolayers at the air-water interface and imaged by Brewster angle microscopy. The ternary monolayers were transferred from the air-water onto the gold electrodes to form bilayer films and were studied for the first time by electrochemical methods: alternative current voltammetry and electrochemical impedance spectroscopy and imaged by atomic force microscopy. In excess of DOPC, the ternary systems remained too liquid for the raft region to be stable, while in the excess of cholesterol the layers were too solid. The layers with SM in excess lead to the formation of Chol:SM complexes but the amount of the fluid matrix was very low. The equimolar content of the three components lead to the formation of a stable and well-organized assembly with well-developed raft microdomains of larger thickness, surrounded by the more fluid part of the bilayer. The latter is proposed as a convenient raft model membrane for further physicochemical studies of interactions with drugs or pollutants or incorporation of membrane proteins.

Incorporation of simvastatin into lipid membranes: Why deliver a statin in form of inclusion complex with hydrophilic cyclodextrin.

In this work, the effects of simvastatin (SIM), (2-hydroxypropyl)-ß-cyclodextrin (HPßCD) and their complex (SIM:HPßCD) on the structure and properties of lipid membranes were investigated for the first time by Langmuir technique combined with PM-IRRAS spectroscopy. An improved understanding of the differences of the interactions between free SIM, and SIM in the form of an inclusion complex with HPßCD with the lipid membrane will improve the development of preparation methods for in vivo applications. Monolayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol (Chol) and their mixture DMPC:Chol (7:3) served as simple models of one leaflet of the cell membrane. The penetration of well-organized lipid layers by simvastatin lead to their fluidization but the extent of this unwanted effect was smaller when the drug was delivered in the form of the SIM:HPßCD complex. Surface pressure vs. time dependencies showed that the drug encapsulated with cyclodextrin dissociated from the complex upon contact with the lipid layer and the weak interactions between the exterior polar part of the HPßCD and the polar headgroups of the lipid layer facilitated smooth incorporation of the released lipophilic drug into the membrane. At a longer time-scale, the HPßCD ligand released from the complex removed some cholesterol, but not DMPC, from the lipid layer, hence, similarly to the enzyme inhibiting action of statins - it lead to the decrease of the amount of cholesterol in the membrane. Delivery of simvastatin in the form of an inclusion complex with HPßCD is proposed as an approach improving its bioavailability in the cholesterol-lowering therapies.

Interactions of anticancer drugs doxorubicin and idarubicin with lipid monolayers: New insight into the composition, structure and morphology.

We quantify directly here for the first time the extents of interactions of two different anthracycline drugs with pure and mixed lipid monolayers with respect to the surface pressure and elucidate differences in the resulting interaction mechanisms. The work concerns interactions of doxorubicin (DOx) and idarubicin (IDA) with monolayers of the zwitterionic DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and negatively charged DMPS (1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt)) as well as a 7:3 mixture of the two lipids. These drugs are used in current cancer treatments, while the lipid systems were chosen as phosphocholines are the major lipid component of healthy cell membranes, and phosphoserines are the major lipid component that is externalized into the outer leaflet of cancerous cell membranes. It is shown that DOx interacts with DMPS monolayers to a greater extent than with DMPC monolayers by lower limits of a factor of 5 at a surface pressure of 10 mN/m and a factor of 12 at 30 mN/m. With increasing surface pressure, the small amount of drug (~0.3 µmol/m2) bound to DMPC monolayers is excluded from the interface, yet its interaction with DMPS monolayers is enhanced until there is even more drug (~3.2 µmol/m2) than lipid (~2.6 µmol/m2) at the interface. Direct evidence is presented for all systems studied that upon surface area compression lipid is reproducibly expelled from the monolayer, which we infer to be in the form of drug-lipid aggregates, yet the nature of adsorption of material back to the monolayer upon expansion is system-dependent. At 30 mN/m, most relevant to human physiology, the interactions of DOx and IDA are starkly different. For DOx, there is a conformational change in the interfacial layer driven by aggregation, resulting in the formation of lateral domains that have extended layers of drug. For the more lipophilic IDA, there is penetration of the drug into the hydrophobic acyl chain region of the monolayer and no indication of lateral segregation. In addition to the Langmuir technique, these advances were made as a result of direct measurements of the interfacial composition, structure and morphology using two different implementations of neutron reflectometry and Brewster angle microscopy. The results provide new insight into key processes that determine the uptake of drugs such as limited drug penetration through cell membranes by passive diffusion as well as activation of drug removal mechanisms related to multidrug resistance.

Lipid membranes exposed to dispersions of phytantriol and monoolein cubosomes: Langmuir monolayer and HeLa cell membrane studies.

The interactions of liquid-crystalline nanoparticles based on lipid-like surfactants, glyceryl monooleate, monoolein (GMO) and 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecane, phytantriol (PT) with selected model lipid membranes prepared by Langmuir technique were compared. Monolayers of DPPC, DMPS and their mixture DPPC:DMPS 87:13 mol% were used as simple models of one leaflet of a cell membrane. The incorporation of cubosomes into the lipid layers spread at the air-water interface was followed by surface-pressure measurements and Brewster angle microscopy. The cubosome - membrane interactions lead to the fluidization of the model membranes but this effect depended on the composition of the model membrane and on the type of cubosomes. The interactions of PT cubosomes with lipid layers, especially DMPS-based monolayer were stronger compared with those of GMO-based nanoparticles. The kinetics of incorporation of cubosomal material into the lipid layer was influenced by the extent of hydration of the polar headgroups of the lipid: faster in the case of smaller, less hydrated polar groups of DMPS than for strongly hydrated uncharged choline of DPPC. The membrane disrupting effect of cubosomes increased at longer times of the lipid membrane exposure to the cubosome solution and at larger carrier concentrations. Langmuir monolayer observations correspond well to results of studies of HeLa cells exposed to cubosomes. The larger toxicity of PT cubosomes was confirmed by MTS. Their ability to disrupt lipid membranes was imaged by confocal microscopy. On the other hand, PT cubosomes easily penetrated cellular membranes and released cargo into various cellular compartments more effectively than GMO-based nanocarriers. Therefore, at low concentrations, they may be further investigated as a promising drug delivery tool.

The influence of charge and lipophilicity of daunorubicin and idarubicin on their penetration of model biological membranes - Langmuir monolayer and electrochemical studies.

The interactions of two selected anthracyclines, daunorubicin (DNR) and idarubicin (IDA), with phospholipid monolayers used as simple models of cell membranes, were investigated. The results of Langmuir experiments together with Brewster angle microscopy showed that both drugs strongly affect cancer cell membranes composed of 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS). Electrostatic interactions allow positively charged DNR and IDA to interact with negatively charged DMPS polar heads but increased lipophilicity of IDA allows it to penetrate the layer more effectively than DNR and prevents from its expulsion at higher surface pressures. The analysis of the thermodynamical functions of hysteresis proves the presence of the enthalpically favorable interactions within the monolayer during its compression in the presence of idarubicin, which may form aggregates with DMPS molecules. The influence of the drugs was significantly less pronounced for a healthy cell model composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) due to the lack of strong electrostatic attractions. The interactions of drugs with pre-compressed phospholipid monolayers were also examined. The physical state of the monolayer and its packing determined only to some extent the penetration of anthracyclines. Since drug molecules first approach the polar region of the monolayer, the increase in surface pressure in time was more pronounced for negatively charged DMPS monolayers than for zwitterionic DMPC. Additionally, idarubicin was able to penetrate the precompressed DMPS monolayers more effectively than daunorubicin due to increased lipophilicity. This property of the drug was also responsible for IDA better penetration of hydrocarbon chains of supported DMPS monolayers compared to DNR, as shown by electrochemical studies.

PM-IRRAS Study on the Effect of Phytantriol-Based Cubosomes on DMPC Bilayers as Model Lipid Membranes.

The effect of phytantriol (PT)-based liquid-crystalline nanoparticles, cubosomes, on the lipid bilayer membranes has been investigated using the combined Langmuir-Blodgett/Langmuir-Schaefer (LB-LS) technique to form an h-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) monolayer at the air-water interface and transfer the lipid bilayer onto the Au(111) substrate. Changes of the compression isotherms confirmed incorporation of cubosomes dispersed in the subphase into the h-DMPC monolayer at the air-water interface. The photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements of the gold electrode covered by the transferred DMPC bilayer showed for the first time how the incorporation of cubosome material affects the orientation and conformation of lipid molecules in the membrane. Exposure to cubosomes affected the packing of d54-DMPC bilayers and introduced disorder of chains by increasing the contribution of gauche conformation. The decrease of the tilt angle of the acyl chains of adsorbed DMPC in the whole range of potentials applied to the gold electrode confirmed that incorporation of cubosome material results in a more tightly packed bilayer. The presence of phytantriol molecules within the d63-DMPC matrix was confirmed by PM-IRRAS studies of the PT-related bands. The LB and PM-IRRAS studies demonstrated in a convincing way that PT-based cubosomes change the organization of model lipid layers leading to structural changes of the membranes which have to be taken into consideration when PT-cubosomes are employed as drug carriers.

Binding of a Myristoylated Protein to the Lipid Membrane Influenced by Interactions with the Polar Head Group Region.

Many cytoplasmic proteins contain a hydrophobic acyl chain, which facilitates protein binding to cell membranes. Hydrophobic interactions between the exposed acyl chain of the protein and hydrocarbon chains of lipids in the cell membrane are the driving force for this specific lipid-protein interaction. Recent studies point out that in addition to hydrophobic interactions the charge-charge and charge-dipole interactions between the polar head groups and basic amino acids contribute significantly to the binding process. Recoverin possesses a myristoyl chain at the N-terminus. In the presence of Ca2+ ions, the protein undergoes structural rearrangements, leading to the extrusion of the myristoyl chain, facilitating the protein binding to the membrane. In this work, we investigate the impact of interactions between the polar head group region of lipid molecules and recoverin which binds to the model membrane. The interaction with a planar lipid bilayer composed of phosphatidylcholine and cholesterol with myristoylated and nonmyristoylated recoverin is studied by in situ polarization modulation infrared reflection absorption spectroscopy. The binding of recoverin to the lipid bilayer depends on the transmembrane potential, indicating that the orientation of the permanent surface dipole in the supramolecular assembly of the lipid membrane influences the protein attachment to the membrane surface. Analysis of the amide I' mode indicates that the orientation of recoverin bound to the lipid bilayer is independent of the presence of myristoyl chain in the protein and of the folding of the protein into the tense or relaxed state. In contrast, it changes as a function of the membrane potential. At positive transmembrane potentials, the α-helical fragments of recoverin are oriented predominantly parallel to the bilayer surface. This orientation facilitates the insertion of the acyl chain of the protein into the hydrophobic region of the bilayer. At negative transmembrane potentials, the α-helical fragments of recoverin change their orientation with respect to the membrane surface, which is followed by the removal of the myristoyl chain from the membrane.

Structure of surfactant and phospholipid monolayers at the air/water interface modeled from neutron reflectivity data.

Specular neutron reflectometry is a powerful technique to resolve interfacial compositions and structures in soft matter. Surprisingly however, even after several decades, a universal modeling approach for the treatment of data of surfactant and phospholipid monolayers at the air/water interface has not yet been established. To address this shortcoming, first a systematic evaluation of the suitability of different models is presented. The result is a comprehensive validation of an optimum model, which is evidently much needed in the field, and which we recommend as a starting point for future data treatment. While its limitations are openly discussed, consequences of failing to take into account various key aspects are critically examined and the systematic errors quantified. On the basis of this physical framework, we go on to show for the first time that neutron reflectometry can be used to quantify directly in situ at the air/water interface the extent of acyl chain compaction of phospholipid monolayers with respect to their phase. The achieved precision of this novel quantification is ∼10%. These advances together enhance significantly the potential for exploitation in future studies data from a broad range of systems including those involving synthetic polymers, proteins, DNA, nanoparticles and drugs.

Synthesis, characterization, and interactions of single-walled carbon nanotubes modified with doxorubicin with Langmuir-Blodgett biomimetic membranes.

The synthesis, characterization, and the influence of single-walled carbon nanotubes (SWCNTs) modified with an anticancer drug doxorubicin (DOx) on the properties of model biological membrane as well as the comparison of the two modes of modification has been presented. The drug was covalently attached to the nanotubes either preferentially on the sides or at the ends of the nanotubes by the formation of hydrazone bond. The efficiency of the modification was proved by the results of FTIR, Raman, and thermogravimetric analysis. In order to characterize the influence of SWCNT-DOx conjugates on model biological membranes, Langmuir technique has been employed. The mixed monolayers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) and SWCNT-DOx with different weight ratio have been prepared. It has been shown that changes in the isotherm characteristics depend on the SWCNTs content. While smaller amounts of SWCNTs do not exert significant differences, the introduction of the prevailing content of the nanotubes increases area per molecule and decreases the maximum value of compression modulus, leading to more fluid monolayer. However, upon increasing the surface pressure, the aggregation of carbon nanotubes within the thiolipid matrix has been observed. Mixed layers of DPPTE/SWCNT-DOx were also transferred onto gold electrodes by means of LB method. Cyclic voltammetry showed that SWCNT-DOx conjugates remain adsorbed at the electrode surface and are stable in time. Additionally, higher values of peak current and DOx surface concentration obtained for side modification prove that side modification allows for more efficient conjugation of the drug to carbon nanotubes. Graphical abstractᅟ.

Interactions of Lipidic Cubic Phase Nanoparticles with Lipid Membranes.

The interactions of liquid-crystalline monoolein (GMO) cubic phase nanoparticles with various model lipid membranes spread at the air-solution interface by the Langmuir technique were investigated. Cubosomes have attracted attention as potential biocompatible drug delivery systems, and thus understanding their mode of interaction with membranes is of special interest. Cubosomes spreading at the air-water interface as well as interactions with a monolayer of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) compressed to different surface pressures were studied by monitoring surface pressure-time dependencies at constant area. Progressive incorporation of the nanoparticles was shown to lead to mixed monolayer formation. The concentration of cubosomes influenced the mechanism of incorporation, as well as the fluidity and permeability of the resulting lipid membranes. Brewster angle microscopy images reflected the dependence of the monolayer structure on the cubosomes presence in the subphase. A parameter Csat was introduced to indicate the point of saturation of the lipid membrane with the cubosomal material. This parameter was found to depend on the surface pressure showing that the cubosomes disintegrate in prolonged contact with the membrane, filling available voids in the lipid membrane. At highest surface pressures when the layer is most compact, the penetration of cubosomal material is not possible and only some exchange with the membrane lipid becomes the route of including GMO into the layer. Finally, comparative studies of the interactions between lipids with various headgroup charges with cubosomes suggest that at high surface pressure an exchange of lipid component between the monolayer and the cubosome in its intact form may occur.

Comparison of the interactions of daunorubicin in a free form and attached to single-walled carbon nanotubes with model lipid membranes.

In this work the interactions of an anticancer drug daunorubicin (DNR) with model thiolipid layers composed of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) were investigated using Langmuir technique. The results obtained for a free drug were compared with the results recorded for DNR attached to SWCNTs as potential drug carrier. Langmuir studies of mixed DPPTE-SWCNTs-DNR monolayers showed that even at the highest investigated content of the nanotubes in the monolayer, the changes in the properties of DPPTE model membranes were not as significant as in case of the incorporation of a free drug, which resulted in a significant increase in the area per molecule and fluidization of the thiolipid layer. The presence of SWCNTs-DNR in the DPPTE monolayer at the air-water interface did not change the organization of the lipid molecules to such extent as the free drug, which may be explained by different types of interactions playing crucial role in these two types of systems. In the case of the interactions of free DNR the electrostatic attraction between positively charged drug and negatively charged DPPTE monolayer play the most important role, while in the case of SWCNTs-DNR adducts the hydrophobic interactions between nanotubes and acyl chains of the lipid seem to be prevailing. Electrochemical studies performed for supported model membranes containing the drug delivered in the two investigated forms revealed that the surface concentration of the drug-nanotube adduct in supported monolayers is comparable to the reported surface concentration of the free DNR incorporated into DPPTE monolayers on gold electrodes. Therefore, it may be concluded that the application of carbon nanotubes as potential DNR carrier allows for the incorporation of comparable amount of the drug into model membranes with simultaneous decrease in the negative changes in the membrane structure and organization, which is an important aspect in terms of side effects of the drug.

PM-IRRAS Studies of DMPC Bilayers Supported on Au(111) Electrodes Modified with Hydrophilic Monolayers of Thioglucose.

A phospholipid bilayer composed of 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine (d54-DMPC) was deposited onto the Au(111) electrode modified with a self-assembled monolayer of 1-thio-ß-d-glucose (ß-Tg) via the Langmuir-Blodgett and Langmuir-Schaefer (LB-LS) techniques. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) measurements were used to characterize structural and orientational changes in this model biological membrane on a hydrophilic surface modified gold electrode. The results of the spectroscopic measurements showed that the tilt angle of acyl chains obtained for deuterated DMPC bilayers supported on the ß-Tg-modified gold is significantly lower than that reported previously for DMPC bilayers deposited directly on Au(111) electrodes. Moreover, tilt angles of ∼18° were obtained for d54-DMPC bilayers on ß-Tg self-assembled monolayers (SAMs) at positive potentials, which are similar to the values calculated for h-DMPC deposited on bare gold in the desorbed state and to those observed for a stack of hydrated DMPC bilayers. This data confirms that the ß-thioglucose SAM promotes the formation of a water cushion that separates the phospholipid bilayer from the metal surface. As a result, the DMPC polar heads are not in direct contact with the electrode and can adopt a zigzag configuration, which strengthens the chain-chain interactions and allows for an overall decrease in the tilt of the acyl chains. These novel supported model membranes may be especially useful in studies pertaining to the incorporation of peptides and proteins into phospholipid bilayers.

pH dependence of daunorubicin interactions with model DMPC:Cholesterol membranes.

Mixed monolayers composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC):Cholesterol 7:3 prepared by the Langmuir-Blodgett method were used as model membranes to investigate the influence of the anticancer drug daunorubicin (DNR) on the properties of the lipid membrane. The Langmuir monolayer experiments revealed that drug - membrane interactions are pH-dependent. The changes in monolayer organization at subphases of pH 7.4 containing daunorubicin visualized by Brewster Angle Microscopy showed that in the presence of the drug the typical morphology observed for phospholipid layers containing cholesterol was no longer seen. It supports the explanation of the mechanism of the drug incorporation into the layers in terms of the competition between DNR molecules and cholesterol in the layer. Increasing surface pressure with time and increasing value of limiting surface pressure with increasing drug concentration in the subphase confirmed incorporation of the drug into the membranes. The interactions between the lipid monolayer and the drug at pH 5.4 were of electrostatic nature between the negative part of the DMPC molecule and positively charged drug, while at pH 7.4 contribution of interactions of daunorubicin with cholesterol was observed. Large differences of the surface-pressure vs. time plots were observed at both pH values when the DMPC:Cholesterol monolayer was not well organized yet. The voltammograms recorded for DMPC:Cholesterol monolayers transferred from the air-water interface onto gold electrode confirmed the presence of the drug in the lipid layer. Based on the charge of the oxidation-reduction peaks corresponding to the redox processes of quinone-hydroquinone group in daunorubicin, the initial surface concentration of the drug in the membrane and the drug release profile to the solution were evaluated.

Dependence of interfacial film organization on lipid molecular structure.

Combination of surface analytical techniques was employed to investigate the interfacial behavior of the two designed lipids-N-stearoylglycine (1) and its bulky neutral headgroup-containing derivative N-stearoylvaline ethyl ester (2)-at the air-solution interface and as transferred layers on different substrates. Formation of monolayers at the air-water interface was monitored on pure water and on aqueous solutions of different pH. Crystallization effects were visualized at pure water by recording the hystereses in the Langmuir-Blodgett (LB) isotherms and by transferring the layers onto mica, gold (111), and ITO (indium-tin oxide on glass) electrodes. Subphase pH affects the morphology and patch formation in monolayers of 1, as evidenced by BAM measurements. At pH 8.2, formation of well-ordered crystallites is observed, which upon compression elongate according to predominantly 1-D growth mechanism to form a dense layer of crystallites. This effect is not observed in monolayers of 2, whose headgroup is not protonated. The orientation of layers of 1 transferred to the solid supports is also pH dependent, and their stability can be related to formation of a hydrogen-bonded networks. AFM images of 1 exhibited platelets of multilayer phase. The IR spectra of the ITO substrates covered by 1 indicated formation of hydrogen bonds between the amide groups. The nature of the adsorption layer and its organization as a function of potential were studied in-depth by EC STM using Au(111) as the substrate. A model showing the arrangement of hydrogen bonds between adsorbed molecules is presented and related to the observed organization of the layer.

Electrochemical and microscopic characteristics of thiolipid layers as simple models of cell membranes.

The thiolipid 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) has been proposed as a component of a simple model of cell membranes, which can be used for the studies of the interactions with drugs and other biologically important species. Depending on the deposition technique, Langmuir-Blodgett method or self-assembly, the obtained model membranes exhibit differences in the organization and properties, as shown by electrochemical techniques. The surface concentration and area per molecule of DPPTE model membranes were determined using chronocoulometry, which gives more reliable results than the widely used reductive desorption method. The mean surface concentration of self-assembled DPPTE monolayer deposited on gold electrode is equal to 4.52 × 10(-10) mol·cm(-2), which corresponds to the area per molecule of 36.7 Å(2). Moreover, model membranes prepared by means of LB method tend to be less compact than self-assembled DPPTE monolayers. Adsorption/desorption behavior of the DPPTE molecules on Au(111) was also visualized by EC-STM method. At the beginning of the process at negative potentials, the physisorbed molecules formed a flat-lying adlayer. Changing the potential in the positive direction resulted in the formation of Au-S bonds, and as a consequence the upstanding phase with higher packing density was observed. The thickness of such a layer determined by atomic force microscopy method is equal to 2.08 nm and corresponds to that of a monomolecular film.