Formation of sparsely tethered bilayer lipid membrane on a biodegradable self-assembled monolayer of poly(lactic acid).

The utilization of biomimetic membranes supported by advanced self-assembled monolayers is gaining attraction as a promising sensing tool. Biomimetic membranes offer exceptional biocompatibility and adsorption capacity upon degradation, transcending their role as mere research instruments to open new avenues in biosensing. This study focused on anchoring a sparsely tethered bilayer lipid membrane onto a self-assembled monolayer composed of a biodegradable polymer, functionalized with poly(ethylene glycol)-cholesterol moieties, for lipid membrane integration. Real-time monitoring via quartz crystal microbalance, coupled with characterization using surface-enhanced infrared absorption spectroscopy and electrochemical impedance spectroscopy, provided comprehensive insights into each manufacturing phase. The resulting lipid layer, along with transmembrane pores formed by gramicidin A, exhibited robust stability. Electrochemical impedance spectroscopy analysis confirmed membrane integrity, successful pore formation, and consistent channel density. Notably, gramicidin A demonstrated sustained functionality as an ion channel upon reconstitution, with its functionality being effectively blocked and inhibited in the presence of calcium ions. These findings mark significant strides in developing intricate biodegradable nanomaterials with promising applications in biomedicine.

Stimuli-Responsive Oligourea Molecular Films.

We have designed and synthesized a helical cysteamine-terminated oligourea foldamer composed of ten urea residues featuring side carboxyl and amine groups. The carboxyl group is located in proximity to the C-terminus of the oligourea and hence at the negative pole of the helix dipole. The amine group is located close to the N-terminus and hence at the positive pole of the helix dipole. Beyond the already remarkable dipole moment inherent in oligourea 2.5 helices, the incorporation of additional charges originating from the carboxylic and amine groups is supposed to impact the overall charge distribution along the molecule. These molecules were self-assembled into monolayers on a gold substrate, allowing us to investigate the influence of an electric field on these polar helices. By applying surface-enhanced infrared reflection-absorption spectroscopy, we proved that molecules within the monolayers tend to reorient themselves more vertically when a negative bias is applied to the surface. It was also found that surface-confined oligourea molecules affected by the external electric field tend to rearrange the electron density at urea groups, leading to the stabilization of the resonance structure with charge transfer character. The presence of the external electric field also affected the nanomechanical properties of the oligourea films, suggesting that molecules also tend to reorient in the ambient environment without an electrolyte solution. Under the same conditions, the helical oligourea displayed a robust piezoresponse, particularly noteworthy given the slender thickness of the monolayer, which measured approximately 1.2 nm. This observation demonstrates that thin molecular films composed of oligoureas may exhibit stimulus-responsive properties. This, in turn, may be used in nanotechnology systems as actuators or functional films, enabling precise control of their thickness in the range of even fractions of nanometers.

Surface-Bioengineered Extracellular Vesicles Seeking Molecular Biotargets in Lung Cancer Cells.

Personalized medicine is a new approach to modern oncology. Here, to facilitate the application of extracellular vesicles (EVs) derived from lung cancer cells as potent advanced therapy medicinal products in lung cancer, the EV membrane was functionalized with a specific ligand for targeting purposes. In this role, the most effective heptapeptide in binding to lung cancer cells (PTHTRWA) was used. The functionalization process of EV surface was performed through the C- or N-terminal end of the heptapeptide. To prove the activity of the EVs functionalized with PTHTRWA, both a model of lipid membrane mimicking normal and cancerous cell membranes as well as human adenocarcinomic alveolar basal epithelial cells (A549) and human normal bronchial epithelial cells (BEAS-2B) have been exposed to these bioconstructs. Magnetic resonance imaging (MRI) showed that the as-bioengineered PTHTRWA-EVs loaded with superparamagnetic iron oxide nanoparticle (SPIO) cargos reach the growing tumor when dosed intravenously in NUDE Balb/c mice bearing A549 cancer. Molecular dynamics (MD) in silico studies elucidated a high affinity of the synthesized peptide to the α5ß1 integrin. Preclinical safety assays did not evidence any cytotoxic or genotoxic effects of the PTHTRWA-bioengineered EVs.

De novo designed self-assembling helicomimetic lipooligoureas with antibacterial activity.

The overuse of antibiotics has led to a rise in infections caused by multidrug-resistant bacteria, resulting in a need for new antibacterial compounds with different modes of action. In this paper, we describe a new class of compounds called lipooligoureas, which are foldamer-based mimetics of antimicrobial lipopeptides. The lipooligoureas consist of an acyl chain connected to the N-terminus of an oligourea head group that exhibits a well-defined 2.5-helix secondary structure, which is further stabilized by the attachment of the lipophilic chain to the oligourea moiety. These compounds meet the established criteria for membranolytic compounds by possessing an amphiphilic structure that promotes the internalization and partitioning of the molecules into the lipid membrane. The presence of positively charged urea residues promotes electrostatic interactions with the negatively charged bacterial membrane. The subtle structural differences in oligourea head group influence the compounds' aggregation behavior, with the number and position of positively charged urea residues correlating with their aggregation ability. The biological activity of these compounds in inhibiting bacterial growth is correlated with their ability to aggregate, with stronger antibacterial properties exhibited by those that aggregate more easily. However, the concentration inhibiting bacterial growth is significantly lower than the critical aggregation concentration values, suggesting that the mechanism of action involves the monomeric forms of lipooligoureas. Nonetheless, a mechanism based on membrane-induced aggregation cannot be ruled out. The lipooligoureas exhibit higher activity towards Gram-positive bacteria than against Gram-negative bacteria, which is indicative of certain selectivity of these compounds. It is also demonstrated that lipooligoureas exhibit increased stability against proteolytic degradation in human blood serum.

Sparsely tethered bilayer lipid membranes formed by self-assembly of bicelles: Spectroelectrochemical characterization and incorporation of transmembrane protein.

Many biochemical processes related to proper homeostasis take place in cell membranes. The key molecules involved in these processes are proteins, including transmembrane proteins. These macromolecules still challenge the understanding of their function within the membrane. Biomimetic models that mimic the properties of the cell membrane can help understand their functionality. Unfortunately, preserving the native protein structure in such systems is problematic. A possible solution to this problem involves the use of bicelles. Their unique properties make integrating bicelles with transmembrane proteins manageable while preserving their native structure. Hitherto, bicelles have not been used as precursors for protein-hosting lipid membranes deposited on solid substrates like pre-modified gold. Here, we demonstrated that bicelles can be self-assembled to form sparsely tethered bilayer lipid membranes and the properties of the resulting membrane satisfy the conditions suitable for transmembrane protein insertion. We showed that the incorporation of α-hemolysin toxin in the lipid membrane leads to a decrease in membrane resistance due to pore formation. Simultaneously, the insertion of the protein causes a drop in the capacitance of the membrane-modified electrode, which can be explained by the dehydration of the polar region of the lipid bilayer and the loss of water from the submembrane region.

New, fast and cheap prediction tests for BRCA1 gene mutations identification in clinical samples.

Despite significant progress in cancer therapy, cancer is still the second cause of mortality in the world. The necessity to make quick therapeutic decisions forces the development of procedures allowing to obtain a reliable result in a quick and unambiguous manner. Currently, detecting predictive mutations, including BRCA1, is the basis for effectively treating advanced breast cancer. Here, we present new insight on gene mutation detection. We propose a cheap BRCA1 mutation detection tests based on the surface plasmon resonance (SPR) or quartz crystal microbalance with energy dissipation (QCM-D) response changes recorded during a hybridization process of an oligonucleotide molecular probe with DNA fragments, with and without the BRCA1 mutation. The changes in the morphology of the formed DNA layer caused by the presence of the mutation were confirmed by atomic force microscopy. The unique property of the developed SPR and QCM tests is really short time of analysis: ca. 6 min for SPR and ca. 25 min for QCM. The proposed tests have been verified on 22 different DNA extracted from blood leukocytes collected from cancer patients: 17 samples from patients with various BRCA1 gene mutation variants including deletion, insertion and missense single-nucleotide and 5 samples from patients without any BRCA1 mutation. Our test is a response to the need of medical diagnostics for a quick, unambiguous test to identify mutations of the BRCA1 gene, including missense single-nucleotide (SNPs).

Brownian Motion Influence on AFM Exosomes' Size Measurements.

Extracellular vesicles are evaluated by nanoparticle tracking analysis (NTA), providing information on their hydrodynamic diameters, and by atomic force microscopy (AFM) to calculate their geometric diameters. The aim of this study is to explore the influence of Brownian movements in a sample drop and preparation time on imaging-based measurements and to determine the relationship between the geometric and hydrodynamic sizes of the extracellular vesicles measured by the AFM and the NTA, respectively. Exosomes derived from the human prostate cancer cell line PC3 were evaluated by NTA and AFM, and those results were compared with Monte Carlo simulations. The mean size, evaluated by AFM shortly after application on the mica substrate, is less than its real value. It obtains the correct value faster for a thinner sample drop. Fitting the log-normal distribution to the geometric and hydrodynamic diameters leads to the conclusion that the latter could arise from the former by linear scaling by a factor that could be used to characterize the analyzed extracellular vesicles. The size of the vesicles attached to the mica substrate depends on time. The effect of Brownian motion and stretch of the lipid bilayer should be considered in the context of exosome AFM studies.

Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS) to Probe Interfacial Water in Floating Bilayer Lipid Membranes (fBLMs).

Floating bilayer lipid membranes (fBLMs) immobilized on metallic surfaces provide a convenient model mimicking the cell membranes due to the effective hydration of lipid polar heads in a proximal leaflet and the possibility to generate the potential gradient across the membrane. This chapter describes the protocol for the measurement of interfacial water separating the floating bilayer lipid membrane from the solid support using surface-enhanced infrared absorption spectroscopy (SEIRAS) under electrochemical control.

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.

Ion-Pairing Mechanism for the Valinomycin-Mediated Transport of Potassium Ions across Phospholipid Bilayers.

The role of the anion on the ionophore properties of valinomycin was studied in a model floating bilayer lipid membrane (fBLM) using supporting electrolytes containing K+ with four different counter anion species (ClO4-, H2PO4-, Cl-, and F-). The electrochemical impedance spectra indicate that the membrane resistance of the bilayer decreases with the decrease of Gibbs free energy of anion solvation. The IR spectra demonstrate that valinomycin does not readily bind to K+ in the KH2PO4, KCl, and KF electrolyte solutions, but in the presence of KClO4, valinomycin readily binds to K+, forming a valinomycin-K+ complex. The results in the present paper reveal the role of the counter anion on the transport of cations by valinomycin across the lipid bilayer. The valinomycin-cation complex creates an ion pair with the anion, and this ion pair can enter the hydrophobic region of the bilayer transporting the cation across the membrane. Anions with low solvation energies facilitate the formation of the ion pair improving the ion conductivity of valinomycin-incorporated bilayers. This paper sheds new light on the transport mechanism of valinomycin ionophores and provides new information about the bioactivity of this molecule.

Interactions of Linear Analogues of Battacin with Negatively Charged Lipid Membranes.

The increasing resistance of bacteria to available antibiotics has stimulated the search for new antimicrobial compounds with less specific mechanisms of action. These include the ability to disrupt the structure of the cell membrane, which in turn leads to its damage. In this context, amphiphilic lipopeptides belong to the class of the compounds which may fulfill this requirement. In this paper, we describe two linear analogues of battacin with modified acyl chains to tune the balance between the hydrophilic and hydrophobic portion of lipopeptides. We demonstrate that both compounds display antimicrobial activity with the lowest values of minimum inhibitory concentrations found for Gram-positive pathogens. Therefore, their mechanism of action was evaluated on a molecular level using model lipid films mimicking the membrane of Gram-positive bacteria. The surface pressure measurements revealed that both lipopeptides show ability to bind and incorporate into the lipid monolayers, resulting in decreased ordering of lipids and membrane fluidization. Atomic force microscopy (AFM) imaging demonstrated that the exposure of the model bilayers to lipopeptides leads to a transition from the ordered gel phase to disordered liquid crystalline phase. This observation was confirmed by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) results, which revealed that lipopeptide action causes a substantial increase in the average tilt angle of lipid acyl chains with respect to the surface normal to compensate for lipopeptide insertion into the membrane. Moreover, the peptide moieties in both molecules do not adopt any well-defined secondary structure upon binding with the lipid membrane. It was also observed that a small difference in the structure of a lipophilic chain, altering the balance between hydrophobic and hydrophilic portion of the molecules, results in different insertion depth of the active compounds.

Electrochemical Properties of Lipid Membranes Self-Assembled from Bicelles.

Supported lipid membranes are widely used platforms which serve as simplified models of cell membranes. Among numerous methods used for preparation of planar lipid films, self-assembly of bicelles appears to be promising strategy. Therefore, in this paper we have examined the mechanism of formation and the electrochemical properties of lipid films deposited onto thioglucose-modified gold electrodes from bicellar mixtures. It was found that adsorption of the bicelles occurs by replacement of interfacial water and it leads to formation of a double bilayer structure on the electrode surface. The resulting lipid assembly contains numerous defects and pinholes which affect the permeability of the membrane for ions and water. Significant improvement in morphology and electrochemical characteristics is achieved upon freeze-thaw treatment of the deposited membrane. The lipid assembly is rearranged to single bilayer configuration with locally occurring patches of the second bilayer, and the number of pinholes is substantially decreased. Electrochemical characterization of the lipid membrane after freeze-thaw treatment demonstrated that its permeability for ions and water is significantly reduced, which was manifested by the relatively high value of the membrane resistance.

Physicochemical and Biological Characterization of Novel Membrane-Active Cationic Lipopeptides with Antimicrobial Properties.

We have designed and synthesized new short lipopeptides composed of tetrapeptide conjugated to fatty acids with different chain lengths. The amino acid sequence of the peptide moiety included d-phenylalanine, two residues of l-2,4-diaminobutyric acid and l-leucine. To explore the possible mechanism of lipopeptide action, we have provided a physicochemical characterization of their interactions with artificial lipid membranes. For this purpose, we have used monolayers and bilayers composed of lipids representative of Gram-negative and Gram-positive bacterial membranes. Using surface pressure measurements and atomic force microscopy, we were able to monitor the changes occurring within the films upon exposure to lipopeptides. Our experiments revealed that all lipopeptides can penetrate the lipid membranes and affect their molecular ordering. The latter results in membrane thinning and fluidization. However, the effect is stronger in the lipid films mimicking Gram-positive bacterial membranes. The results of the physicochemical characterization were compared with the biological activity of lipopeptides. The effect of lipopeptides on bacterial growth was tested on several strains of bacteria. It was revealed that lipopeptides show stronger antimicrobial activity against Gram-positive bacteria. At the same time, all tested compounds display relatively low hemolytic activity.

Physicochemical Characterization of Daptomycin Interaction with Negatively Charged Lipid Membranes.

Daptomycin is known as an effective antibiotic lipopeptide which shows activity against the number of Gram-positive pathogens. Its primary target is the bacterial cell membrane. However, the detailed mechanism of daptomycin action is still subject to debate. In this paper, we have investigated the interactions between lipopeptide and model lipid films composed of negatively charged phosphatidylglycerols and cardiolipin. In order to evaluate the effect of daptomycin on the molecular organization and the properties of lipid assemblies, we have used surface pressure measurements and electrochemical methods combined with atomic force microscopy, quartz crystal microbalance, and surface-enhanced infrared absorption spectroscopy. Our results indicate that daptomycin interaction with the lipid membrane is complex. It involves daptomycin aggregation and partial insertion, which in turn affect the charge distribution on both sides of the membrane and may result in a gradient of water chemical potential. The latter can drive the flux of water across the membrane.

The shape of lipid molecules affects potential-driven molecular-scale rearrangements in model cell membranes on electrodes.

Planar asymmetric lipid bilayers composed of phosphatidylethanolamine and phosphatidylglycerol lipids are transferred onto a gold electrode surface. Lipids containing two saturated, one monounsaturated and two monounsaturated hydrocarbon chains compose the model membranes. Results of electrochemically controlled polarization modulation infrared reflection absorption spectroscopy and quartz crystal microbalance with energy dissipation studies reveal two different types of electric potential-dependent structural rearrangements in the bilayers. They are correlated with the geometry of the lipid molecule. Packing parameter correlates the cross-section area of the hydrophobic and hydrophilic parts of amphiphilic molecules. In bilayers composed of lipids with the packing parameter <1, the hydrocarbon chains are tilted with respect to the bilayer plane and the polar head groups are well hydrated. At a threshold potential an abrupt flow of water through the bilayer is connected with membrane dehydration and upward orientation of the chains. In bilayers composed of lipids with packing parameter ≥1, electric potentials have negligible effect on the membrane structure. A simple rule correlating the packing parameter with molecular scale changes occurring at electrified membranes has a large diagnostic implication for biomimetic studies and our understanding of molecular processes occurring in biological cell membranes.

Water Structure in the Submembrane Region of a Floating Lipid Bilayer: The Effect of an Ion Channel Formation and the Channel Blocker.

The structure of water in the submembrane region of the bilayer of DPhPC floating (fBLM) on a monolayer of 1-thio-ß-d-glucose (ß-Tg)-modified gold nanoparticle film was studied by the surface-enhanced infrared absorption spectroscopy (SEIRAS). SEIRAS employs surface enhancement of the mean square electric field of the photon, which is acting on a few molecular layers above the film of gold nanoparticles. Therefore, it is uniquely suited to probe water molecules in the submembrane region and provides unique information concerning the structure of the hydrogen bond network of water surrounding the lipid bilayer. The IR spectra indicated that water with a strong hydrogen network is separating the membrane from the gold surface. This water is more ordered than the water in the bulk. When alamethicin, a peptide forming ion channels, is inserted into the membrane, the network is only slightly loosened. The addition of amiloride, an ion channel blocker, results in a significant decrease in the amount of water in the submembrane region. The remaining water has a significantly distorted hydrogen bond network. This study provides unique information about the effect of the ion channel on water transport across the bilayer. The electrode potential has a relatively small effect on water structure in the submembrane region. However, the IR studies demonstrated that water is less ordered at positive transmembrane potentials. The present results provide significant insight into the nature of hydration of a floating lipid bilayer on the gold electrode surface.

Assessment of Proteus mirabilis Antigen Immunological Complexes by Atomic Force Microscopy.

Atomic force microscopy (AFM) is being increasingly used to directly measure protein interactions in nearly physiological environments. Here, protocols for atomic force microscopy (AFM) for visualization of antigen-antibody complexes are presented. The technique is used to demonstrate complexes formed by rheumatoid arthritis patient antibodies with lipopolysaccharide (LPS) isolated from P. mirabilis (O3) strain S1959 and a synthetic antigen (LPS epitope of 6 N-alpha-(D-galacturonoyl)-L-lysine residues).

Effect of Interfacial Water on the Nanomechanical Properties of Negatively Charged Floating Bilayers Supported on Gold Electrodes.

Floating lipid bilayers composed of phosphatidylglycerols and cardiolipin were deposited on gold electrodes premodified with 1-thio-ß-d-glucose monolayer by spreading of small unilamellar vesicles. The resulting lipid membrane was homogeneous, and its thickness was ∼5.0 nm. Electrochemical characterization combined with surface-enhanced infrared absorption spectroscopy revealed that negative polarization of the electrode leads to accumulation of water molecules in the interfacial region between lipid membrane and the thioglucose film. Moreover, the buildup of water layer was demonstrated to affect the nanomechanical properties of the membrane. The latter was manifested by well-pronounced decrease of Young's modulus of the lipid bilayer correlating with increasing hydration. This effect was ascribed to the decoupling of the membrane from supporting thioglucose film due to the accumulation of interfacial water. As a result, the effective stiffness of the supporting layer is lower and it alters the nanomechanical behavior of lipid membrane. Our results provide strong experimental proof for the correlation between elastic properties of floating lipid membrane and the amount of water accumulated in the submembrane region.

Interactions of bacteriophage T4 adhesin with selected lipopolysaccharides studied using atomic force microscopy.

The interaction between the T4 bacteriophage gp37 adhesin and the bacterial lipopolysaccharide (LPS) is a well-studied system, however, the affinity and strength of the interaction haven't been analyzed so far. Here, we use atomic force microscopy to determine the strength of the interaction between the adhesin and its receptor, namely LPS taken from a wild strain of E. coli B. As negative controls we used LPSs of E. coli O111:B and Hafnia alvei. To study the interaction an AFM tip modified with the gp37 adhesin was used to scan surfaces of mica covered with one of the three different LPSs. Using the correlation between the surface topography images and the tip-surface interaction we could verify the binding between the specific LPS and the tip in contrast to the very weak interaction between the tip and the non-binding LPSs. Using force spectroscopy we could then measure the binding strength by pulling on the AFM tip until it lifted off from the surface. The force necessary to break the interaction between gp37 and LPS from E. coli B, LPS from E. coli O111:B and LPS from H. alvei were measured to be 70 ± 29 pN, 46 ± 13 pN and 45 ± 14 pN, respectively. The latter values are likely partially due to non-specific interaction between the gp37 and the solid surface, as LPS from E. coli O111:B and LPS from H. alvei have been shown to not bind to gp37, which is confirmed by the low correlation between binding and topography for these samples.

Influence of amine and thiol modifications at the 3' ends of single stranded DNA molecules on their adsorption on gold surface and the efficiency of their hybridization.

Adsorption of molecules of DNA (deoxyribonucleic acid) or modified DNA on gold surfaces is often the first step in construction of many various biosensors, including biosensors for detection of DNA with a particular sequence. In this work we study the influence of amine and thiol modifications at the 3' ends of single stranded DNA (ssDNA) molecules on their adsorption on the surface of gold substrates and on the efficiency of hybridization of immobilized DNA with the complementary single stranded DNA. The characterization of formed layers has been carried out using infrared spectroscopy and atomic force microscopy. As model single stranded DNA we used DNA containing 20 adenine bases, whereas the complementary DNA contained 20 thymine bases. We found that the bands in polarization modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS) spectra of layers formed from thiol-modified DNA are significantly narrower and sharper, indicating their higher regularity in the orientation of DNA on gold surface when using thiol linker. Also, hybridization of the layer of thiol-modified DNA containing 20 adenine bases with the respective DNA containing thymine bases leads to formation of much more organized structures than in the case of unmodified DNA or DNA with the amine linker. We conclude that the thiol-modified ssDNA is more promising for the preparation of biosensors, in comparison with the amine-modified or unmodified ssDNA. We have also found that the above-mentioned modifications at the 3' end of ssDNA significantly influence the IR spectrum (and hence the structure) of polycrystalline films formed from such compounds, even though adsorbed fragments contain less than 5% of the DNA chain. This effect should be taken into account when comparing IR spectra of various polycrystalline films formed from modified and unmodified DNA.