Organization Of Monolayers Research Articles

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.

Are Plant-Based Carbohydrate Nanoparticles Safe for Inhalation? Investigating Their Interactions with the Pulmonary Surfactant Using Langmuir Monolayers.

Nanoparticle carriers show promise for drug delivery, including by inhalation, where the first barrier for uptake in the lungs is the monolayer pulmonary surfactant membrane that coats the air/alveoli interface and is critical to breathing. It is imperative to establish the fate of potential nanocarriers and their effects on the biophysical properties of the pulmonary surfactant. To this end, the impact of the nanoparticle surface charge on the lateral organization, thickness, and recompressibility of Langmuir monolayers of model phospholipid-only and phospholipid-protein mixtures was investigated using native and modified forms of nanophytoglycogen, a carbohydrate-based dendritic polymer extracted from corn as monodisperse nanoparticles. We show that the native (quasi-neutral) and anionic nanophytoglycogens have little impact on the phase behavior and film properties. By contrast, cationic nanophytoglycogen alters the film morphology and increases the hysteresis associated with the work of breathing due to its electrostatic interaction with the anionic phospholipids in the model systems. These findings specifically highlight the importance of surface charge as a selection criterion for inhaled nanoformulations.

Effect of lipopolymer (DSPE-PEG750) on phospholipid monolayers and bilayers differing in the structure of the polar head group

Phospholipid liposomes are the most popular kind of vesicles used in drug delivery due to their high biocompatibility with biomembranes. However, this type of liposomes show some limitations related with their short half-life in bloodstream. This problem can be resolved by the coating of their surface with the application of PEG molecules which form steric and entropic barrier, avoiding recognition of encapsulated liposomes by the reticuloendothelial system (RES). Such modification of liposomes significantly affects the physicochemical characteristics of PEGylated liposomes, which is related to the intermolecular interactions between the lipopolymer molecules and the phospholipid that builds the liposome membrane. In our work we investigated the influence of the PEG-ylated lipid (DSPE-PEG750) on physicochemical properties of phospholipid monolayers and bilayers with different structure of the polar group (DPPC, DPPE, DPPS). The phospholipid monolayers were examined with the application of Langmuir monolayer technique and Brewster angle microscopy (BAM) as well as grazing incidence x-ray diffraction (GIXD). The studies performed on phospholipid bilayer systems were related to dynamic light scattering and zeta potential measurements and the experiments with the calcein release and steady-state fluorescence anisotropy of DPH. The obtained results showed that the type of polar group of phospholipid as well as the addition of PEG-ylated lipid significantly changes the molecular organization of phospholipid monolayers. Moreover, these parameters also influence the properties of phospholipid bilayers such as size, surface charge, stability and permeability.

Effect of cavitation intensity control on self-assembling of alkanethiols on gold in room temperature ionic liquids

This study investigates the effect of cavitation intensity on self-assembling of alkanethiol molecules on gold in room temperature ionic liquids (RTILs) under low frequency ultrasound irradiation (20 kHz). The use of RTILs, with low vapor pressure, enabled cavitation activity to be controlled up to quenching through pressure decrease within an argon-saturated atmosphere. This control possibility was used to acquire deeper insights into the role of cavitation on self-assembling processes. It was shown by electrochemical, contact angles and Polarization Modulation - Infrared Reflection Absorption Spectroscopy (PM-IRRAS) measurements that cavitation activates orientation and organization of self-assembled monolayers (SAM). X-ray Photoelectron Spectroscopy (XPS) revealed that, even if chemical adsorption of molecules is highly activated under ultrasound irradiation, it is not dependent on acoustic cavitation intensity.

An electro-conductive hybrid scaffold as an artificial Bruch's membrane

Many research groups have investigated the various kinds of scaffolds to mimic the natural Bruch's membrane (BM) and support the retinal pigmented epithelial cells to form an organized cellular monolayer. While using prosthetic BM is identified as a promising treatment of age-related macular degeneration (AMD), a degenerative and progressive retinal disease, the effects of different signals such as electrical and morphological cues on the retinal pigmented epithelial (RPE) cells are still unknown. In this study, a laminated and conductive hydrogel/fiber composite scaffold by adding conductive polyaniline (PANi) to the scaffold's nanofibrous phase was prepared. This hybrid scaffold offers the closest morphology to the native structure of the human Bruch's membrane by imitating the inner and outer collagenous layer and induces the electrical signal to the scaffold to assess the electrical cue on behaviors of polarized retinal pigmented epithelial cells in the retina. The electrospun nanofibrous phase consisted of gelatin-Polyaniline in different ratios incorporated into the hydrogel precursor, a blend of gelatin and 4-armed PEG. We used a novel dual crosslinking process by incorporating the exposure of gamma irradiation and glutaraldehyde vapor treatment to construct the scaffold's hydrogel phase. The results showed the best composition was the sample which included the 40/60, Polyaniline/gelatin nanofiber sheets ratio because this scaffold revealed a 2.66 ± 0.33 MPa, Young's modulus and 1.84 ± 0.21 S/cm, electrochemical conductivity, which are close to the main features of native Bruch's membrane. In addition, this scaffold showed good biocompatibility by reaching 83.47% cell viability.

Mechanobiology of Epithelia From the Perspective of Extracellular Matrix Heterogeneity.

Understanding the complexity of the extracellular matrix (ECM) and its variability is a necessary step on the way to engineering functional (bio)materials that serve their respective purposes while relying on cell adhesion. Upon adhesion, cells receive messages which contain both biochemical and mechanical information. The main focus of mechanobiology lies in investigating the role of this mechanical coordination in regulating cellular behavior. In recent years, this focus has been additionally shifted toward cell collectives and the understanding of their behavior as a whole mechanical continuum. Collective cell phenomena very much apply to epithelia which are either simple cell-sheets or more complex three-dimensional structures. Researchers have been mostly using the organization of monolayers to observe their collective behavior in well-defined experimental setups in vitro. Nevertheless, recent studies have also reported the impact of ECM remodeling on epithelial morphogenesis in vivo. These new concepts, combined with the knowledge of ECM biochemical complexity are of key importance for engineering new interactive materials to support both epithelial remodeling and homeostasis. In this review, we summarize the structure and heterogeneity of the ECM before discussing its impact on the epithelial mechanobiology.

Impeded Molecular Reorganization by Polyethylene Glycol Conjugation Revealed by X-ray Reflectivity and Diffraction Measurements.

Polyethylene glycol (PEG) coatings have been widely applied in pharmaceutical and biomedical systems to prevent nonspecific protein absorption, increase vesicle blood circulation time, and sustain drug release. This study systematically investigated the planar interfacial organization of phospholipid monolayers containing various amounts of PEG conjugations before and after enzyme-catalyzed degradation of the lipids using X-ray reflectivity and grazing incidence X-ray diffraction techniques. Results showed that attaching PEG to the headgroup of the lipids up to 15 mol % had limited effects on molecular packing of the lipid monolayers in the condensed phase at the gas-liquid interface and negligible effects on the enzyme adsorption to the interface. After enzyme-catalyzed degradation, equimolar fatty acids and lyso PC were generated. The fatty acids together with the subphase Ca2+ self-assembled into highly organized multilayer domains at the interface. The X-ray measurements unambiguously revealed that the densely packed PEG markedly hindered microphase separation and formation of the palmitic acid-Ca2+ complexes.

X-Ray Reflectivity and Diffraction Studies of Doxorubicin Binding to Model Lipid Membranes

The effect of anticancer antibiotic doxorubicin on structural organization of anionic lipid monolayers has been studied. X-ray reflectivity and grazing incidence diffraction techniques were applied to monitor the changes in 2D structure and electron density distribution of Langmuir monolayer composed of negatively charged dipalmitoylphosphatidylglycerol (DPPG) and dioleoylphosphatidylserine (DOPS). For comparison, monolayer of zwitterionic dipalmitoylphosphatidylethanolamine (DPPE) also was investigated. The presented experimental results suggest that doxorubicin interaction with anionic lipid monolayers (DPPG and DOPS) proceeds preferentially via electrostatic attraction—positively charged amino groups of doxorubicin bind to negatively charged head groups of phospholipid molecules. Based on the obtained data, the penetration of doxorubicin into the hydrophobic part of anionic lipid monolayers does not occur. X-ray measurements on DPPE monolayer indicated that doxorubicin did not cause any significant alterations of molecular packing in condensed monolayer of zwitterionic DPPE molecules.

Spontaneous collapse of palmitic acid films on an alkaline buffer containing calcium ions

Understanding the interaction of ions with fatty acids is important to identify their roles in various bioprocesses and to build novel biomimetic systems. In this study, the molecular organization of palmitic acid (PA) films on alkaline buffer solutions (pH 7.4) with and without divalent Ca2+ was measured at a constant surface area using Langmuir troughs coupled with microscopy and X-ray interfacial techniques. Without Ca2+, PA molecules remained a monolayer organization; however, with Ca2+, formation of the inverted bilayers of PA-Ca2+ superstructures caused a spontaneous 2D to 3D transformation under no compression due to the strong interaction between PA and the divalent cation. Self-assembly of this highly-organized inverted bilayer superstructure involved a two-step process of nucleation and nuclei growth. During nucleation, densely packed PA and Ca2+ monolayer firstly corrugated and some of PA and Ca2+ molecules ejected out from the monolayer; the ejected molecules then reorganized and formed the inverted bilayer nuclei. Nucleation was followed by nuclei growth, during which PA and Ca2+ in the monolayer kept integrating into the inverted bilayer structure through molecule migration and PA rotation around Ca2+.

Molecular Features for Probing Small Amphiphilic Molecules with Self-Assembled Monolayer-Protected Nanoparticles.

The sensing of small molecules poses the challenge of developing devices able to discriminate between compounds that may be structurally very similar. Here, attention has been paid to the use of self-assembled monolayer (SAM)-protected gold nanoparticles since they enable a modular approach to tune single-molecule affinity and selectivity simply by changing functional moieties (i.e., covering ligands), along with multivalent molecular recognition. To date, the discovery of monolayers suitable for a specific molecular target has relied on trial-and-error approaches, with ligand chemistry being the main criterion used to modulate selectivity and sensitivity. By using molecular dynamics, we showcase that either individual molecular characteristics and/or collective features such as ligand flexibility, monolayer organization, ligand local ordering, and interfacial solvent properties can also be exploited conveniently. The knowledge of the molecular mechanisms that drive the recognition of small molecules on SAM-covered nanoparticles will critically expand our ability to manipulate and control such supramolecular systems.

Differential impact of synthetic antitumor lipid drugs on the membrane organization of phosphatidic acid and diacylglycerol monolayers

Anti-tumour lipids are synthetic analogues of lysophosphatidylcholine. These drugs are both cytotoxic and cytostatic, and more interestingly, exert these effects preferentially in tumour cells. While the exact mechanism of action isn’t fully elucidated, these drugs appear to preferentially partition into rigid lipid domains in cell membranes. Upon insertion, the compounds alter membrane domain organization, disrupt normal signal transduction, and cause cell death. Recently, it has been reported that these drugs induce accumulation of diacylglycerol in yeast cells which in turn sensitizes cells to the drugs. Conversely, phosphatidic acid accumulation appears to protect cells against the drugs. In the current work, the aim was to compare the biophysical effects of the drugs edelfosine, miltefosine and perifosine on monolayers of dimyristoyl phosphatidic acid, dimyristoyl glycerol and an equimolar mixture, to understand how these lipids modulate the mode of action. Surface pressure – area isotherms, compression moduli and Brewster angle microscopy were used to compare drug effects on lipid packing, monolayer compressibility and lateral domain organization of these films. Results suggest that edelfosine and miltefosine have stabilizing effects on all of the monolayers, while perifosine destabilizes dimyristoyl glycerol and the equimolar mixture. Additionally, all three drugs change the morphology of the domains observed. Based on these results the stabilization of diacylgylcerol by edelfosine and miltefosine may contribute to the mode of action as diacylglycerol is a known disruptor of bilayers. Perifosine however does not stabilize diacylglycerol, and therefore cell death may occur through a more direct inhibition of specific signal transduction. These results suggest that perifosine may illicit cytotoxicity through a different mechanism compared to the other antitumor lipid drugs.

Sustained and improved enzymatic activity of trypsin immobilized in the Langmuir Blodgett film of DPPC: A rapid enzyme sensor for the detection of Azocasein

Immobilizing enzymes on biomimicking monolayer ultrathin films of phospholipids exhibit enhanced enzymatic activities (EAs) useful for biosensing applications. Preparation of such substrates with enzyme Trypsin (TRYP) immobilized in dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) matrix with enhanced and sustained enzymatic activity is a real challenge, as the isoelectric point of TRYP is reported to exist at pH ~ 10.5. This paper reports for the first time a facile procedure to fabricate smart substrates of TRYP entrapped in the DPPC monolayer using Langmuir–Blodgett (LB) technique. The incorporation of TRYP into DPPC monolayer at the air–water interface has been demonstrated from adsorption kinetics (π – t), room temperature surface pressure–area (π – A) isotherm plot and compressibility (β – π) studies. Circular dichroism (CD) and Grazing angle IR (GAIR) spectra signify changes in the secondary structure of incubated TRYP when immobilized in DPPC monolayer. Improved and sustained EA of TRYP upon its incubation on the organized monolayer LB films of DPPC has been observed. The reason behind such improvement in the EA of immobilized TRYP has also been suggested. The as fabricated substrates of immobilized TRYP can be used to sense soy milk, soy yoghurt, mayonnaise, meringue etc. using Azocasein as the protease substrate.

Functionalization of LC molecular films with nanocrystalline cellulose: A study of the self-assembly processes and molecular stability

The bottom-up approach in designing and synthesis of materials using nanoparticles of different size-, shape- or chemical composition is an extreme challenge. However, in this way, new task-specific or general use nanocomposites can be obtained. This work presents, the investigations of the preparation procedures and molecular stability of Langmuir films based on cellulose nanocrystals (CNC) and different liquid crystals. The CNC-based nanocomposite with the 5CB, 5OCB, 5FCB, and 5PCH liquid crystals, have been obtained by the modified Langmuir technique. The investigations of the Langmuir monolayers gives an excellent model system for studying intermolecular interactions at the interface, which allows analyzing the ordering, sorption, desorption, association and phase separation phenomena determining physical and chemical properties of studied systems. The process of self-assembly and molecular organization on the air-liquid interphase was studied for different compositions of LC/CNC films. The compressibility profiles and the mean molecular area have been calculated. The stability of prepared monolayers has been studied to investigate the sorption and desorption processes. The morphology of the prepared systems was determined by Brewster Angle Microscopy (BAM), and the influence of the CNC on LC organization in molecular monolayers on the air-liquid interphase has been studied.

A simple method for the isolation and detailed characterization of primary human proximal tubule cells for renal replacement therapy.

The main physiological functions of renal proximal tubule cells in vivo are reabsorption of essential nutrients from the glomerular filtrate and secretion of waste products and xenobiotics into urine. Currently, there are several established cell lines of human origin available as in vitro models of proximal tubule. However, these cells appeared to be limited in their biological relevance, because essential characteristics of the original tissue are lost once the cells are cultured. As a consequence of these limitations, primary human proximal tubule cells constitute a suitable and a biologically more relevant in vitro model to study this specific segment of the nephron and therefore, these cells can play an important role in renal regenerative medicine applications. Here, we describe a protocol to isolate proximal tubule cells from human nephrectomies. We explain the steps performed for an in-depth characterization of the cells, including the study of markers from others segments of the nephron, with the goal to determine the purity of the culture and the stability of proteins, enzymes, and transporters along time. The human proximal tubule cells isolated and used throughout this study showed many proximal tubule characteristics, including monolayer organization, cell polarization with the expression of tight junctions and primary cilia, expression of proximal tubule-specific proteins, such as megalin and sodium/glucose cotransporter 2, among others. The cells also expressed enzymatic activity for dipeptidyl peptidase IV, as well as for gamma glutamyl transferase 1, and expressed transporter activity for organic anion transporter 1, P-glycoprotein, multidrug resistance proteins, and breast cancer resistance protein. In conclusion, characterization of our cells confirmed presence of putative proximal tubule markers and the functional expression of multiple endogenous organic ion transporters mimicking renal reabsorption and excretion. These findings can constitute a valuable tool in the development of bioartificial kidney devices.

All-Aqueous Thin-Film-Flow-Induced Cell-Based Monolayers.

Cells in vitro usually require a solid scaffold to attach and form two-dimensional monolayer structures. To obtain a substrate-free cell monolayer, long culture time and specific detaching procedures are required. In this study, a thin-film-flow-induced strategy is reported to overcome the challenges of assembling in vitro scaffold-free monolayered cell aggregates. The assembly is driven by a dewetting-like thin-film withdrawal along all-aqueous interfaces characterized by a low interfacial tension. The withdrawal process drives the cells adsorbed on the liquid film to aggregate and assemble into an organized and compact monolayer. This strategy is not limited to biological cells but also colloidal particles, as demonstrated by the assembly of hybrid cell-particle monolayers. The versatility offered by this approach suggests new opportunities in understanding early tissue formation and functionalizing cell monolayer aggregates by colloidal particles with customized functions.

Licofelone-DPPC Interactions: Putting Membrane Lipids on the Radar of Drug Development.

(1) Background: Membrane lipids have been disregarded in drug development throughout the years. Recently, they gained attention in drug design as targets, but they are still disregarded in the latter stages. Thus, this study aims to highlight the relevance of considering membrane lipids in the preclinical phase of drug development. (2) Methods: The interactions of a drug candidate for clinical use (licofelone) with a membrane model system made of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were evaluated by combining Langmuir isotherms, Brewster angle microscopy (BAM), polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS), and grazing-incidence X-ray diffraction (GIXD) measurements. (3) Results: Licofelone caused the expansion of the DPPC isotherm without changing the lipid phase transition profile. Moreover, licofelone induced the reduction of DPPC packing density, while increasing the local order of the DPPC acyl chains. (4) Conclusions: The licofelone-induced alterations in the structural organization of phosphatidylcholine monolayers may be related to its pharmacological actions. Thus, the combination of studying drug-membrane interactions with the pharmacological characterization that occurs in the preclinical stage may gather additional information about the mechanisms of action and toxicity of drug candidates. Ultimately, the addition of this innovative step shall improve the success rate of drug development.

A dithiocarbamate anchoring group as a flexible platform for interface engineering.

The molecular organization and electronic properties of dithiocarbamate (DTC) anchored self-assembled monolayers (SAMs) linked to Au(111) substrates are studied by a combination of X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, and state-of-the-art density functional theory calculations. For that, several piperidine/piperazine precursors with different architecture and substitution patterns are selected. The presented data show that the DTC anchor provides a useful building block for monomolecular self-assembly on coinage metals with both sulfur atoms bonded to the substrate in a way similar to what is usually observed for the more commonly applied thiolate docking group. The combination of the DTC group with the quite flexible piperidine/piperazine cyclic linkers results in a dense molecular packing with an upright orientation of the terminal moieties. The latter comprise phenyl rings bearing various substituents, which enables tuning the interfacial dipole over a wide range. Simulations on two prototypical DTC-docked SAMs help to better understand the experimental observations and provide insight into the local origin of the SAM-induced shifts in the electrostatic energy. In particular, a comparison of measured and simulated XP spectra reveals the significant contribution of the DTC group to the interfacial dipole.

Structural analysis of helicene molecules adsorbed on symmetric surfaces.

Helicenes are chiral polyaromatic hydrocarbon molecules which self-assemble into ordered monolayers on solid substrates, and are of current interest in the study of supramolecular systems and the development of smart materials. In this work we investigate the geometry of helicene monomers and stacked dimers on (111) facets of coinage metals. The geometry of the adsorbed molecules is shaped by the coupling of intermolecular dispersive forces, intramolecular steric repulsion between end rings and surface-molecule interactions. Thus, binding and stereospecificity outcomes vary broadly depending on the identity of molecule/surface pair. Overall, homochiral interactions are found to be more effective than heterochiral stacking, due to a better fit between the helical structures in like dimers. On a surface, this effect is enhanced by the flattening of surface-proximal molecular rings. However, our results show that the "sandwich" effect of the second molecular layer increases molecular footprints in the first layer, with potentially large implications in monolayer organization and surface commensuration.

Sphingosine-1-phosphate protects against brain microvascular endothelial junctional protein disorganization and barrier dysfunction caused by alcohol.

S1P has known endothelial barrier-protective properties, but whether this extends to the BBB is unclear. We hypothesized that alcohol-induced disruption of brain microvascular endothelial barrier function and junctional protein organization can be ameliorated by S1P treatment. Cultured primary HBMEC monolayers were used to characterize endothelial-specific mechanisms of BBB regulation. TER and apparent permeability coefficients for albumin, dextran-4 kDa, and sodium fluorescein were used as indices of barrier function. Junctional localization of Claudin-5, VE-cadherin, and β-catenin was determined by immunofluorescence confocal microscopy. S1P was applied following treatment with alcohol. Alcohol significantly impaired HBMEC TER. Application of S1P after alcohol treatment resulted in a hastened recovery to the baseline HBMEC TER. Alcohol-treated HBMEC had a significantly higher mean permeability than control that was reversed by S1P. Alcohol caused the formation of gaps between cells. Treatment with S1P (after alcohol) increased junctional localization of VE-Cadherin, Claudin-5, and β-catenin. Alcohol impairs the barrier function and junctional organization of HBMEC monolayers. S1P enhanced barrier function and restored junctions in the presence of alcohol, and thus may be useful for restoring BBB function during alcohol intoxication.

Effect of polyols on the DMPC lipid monolayers and bilayers

In this study, the effect of polyols, erythritol, xylitol, mannitol, on a model membrane systems composed of DMPC was investigated using differential scanning calorimetry and Fourier transform infrared spectroscopy. Generally, it is considered that polyols possess strong hydrophilic properties, and either does not interact with the hydrophobic environment at all, or these interactions are very weak. To better understand the mutual interactions between polyols and the lipid system, the Langmuir technique was used to examine the molecular organization of monolayers and to calculate their thickness in the presence of polyols at the subphase. The detailed description of the interactions between polyols and DMPC molecules was complemented by the analysis of the morphology of monolayers with the application of Brewster angle microscopy. From ATR FTIR, the significant spectral shift is observed only for the PO2− stretching band, which correlates strongly with the polyol chain-length. The longer the polyol chain, the weaker the observed interactions with lipid molecules. The most important findings, obtained from thickness measurements, reveal that short-chain polyols may prevent the formation of bilayers by the DMPC molecules under high surface pressure. The changes in the organization of DMPC monolayers on the surface, as visualized by Brewster angle microscopy, showed that the domains observed for phospholipid film spread on pure water differ substantially from those containing polyols in the subphase.