Solid-supported Lipid Bilayers Research Articles

Exploring the Interaction of Lipid Bilayers with Curcumin-Laponite Nanoparticles: Implications for Drug Delivery and Therapeutic Applications.

Curcumin, the active compound in turmeric, is renowned for its anti-inflammatory, antioxidant, and antimicrobial properties, making it beneficial for treating conditions like arthritis, neurodegenerative diseases, and various cancers. Despite its promising therapeutic potential, curcumin's poor bioavailability-due to its rapid metabolism and low solubility-limits its clinical efficacy. To address this, recent research has focused on enhancing curcumin delivery using nanoparticles, liposomes, and novel nanomaterials. Among these, laponite, a synthetic nanoclay, has shown promise in improving curcumin delivery due to its unique properties, including large surface area, dual charge, and stability in solution. This study explores the use of curcumin-laponite nanoparticles as carrier vehicles for controlled delivery to in vitro model membranes. Utilizing advanced techniques such as neutron reflectometry, atomic force microscopy, quartz crystal microbalance with dissipation, and infrared spectroscopy, the interaction between curcumin-laponite nanoparticles and solid-supported lipid bilayers is monitored, revealing enhanced stability and controlled release of curcumin across the membrane. These findings pave the way for the development of curcumin-based therapies targeting cardiovascular, neurological, and oncological diseases, leveraging the synergistic effects of curcumin's biological activity and laponite's delivery capabilities.

Capillary-Assisted Assembly of Polymer Gel-Supported Lipid Bilayers.

The polymer-supported lipid bilayer represents an attractive supramolecular assembly in numerous biophysical and bioanalytical applications. The assembly of polymer-supported membranes with a polymer layer thickness of just a few nanometers is now well-established, but bilayer properties in such a membrane architecture are still influenced by the nearby solid substrate. Polymer-supported lipid bilayer systems with a several micrometers thick polymer layer will overcome this shortcoming. However, formation of a fluid lipid bilayer on a fully hydrated, micrometer thick polymer film using traditional methods (e.g., vesicle fusion and lipid monolayer deposition techniques) remains a challenging task due to the rather unfavorable interfacial conditions for bilayer formation in such a system. Here, we report for the first time on the facile capillary-assisted formation of a lipid bilayer on the surface of a fully hydrated, several micrometers thick polyacrylamide (PAA) gel, in which forced molecular crowding of lipids at the air-water interface of the capillary results in monolayer instability and collapse, thereby forming a lipid bilayer on the top of the polymer gel inside the capillary. Stable bilayer attachment on the surface of the polymer gel can be achieved via physisorption or specific chemical linkages (tethering) on both cross-linked and non-cross-linked PAA films. Unlike the traditional solid-supported lipid bilayer (SLB), the lipid lateral diffusion in the polymer gel-supported lipid bilayer is not anymore perturbed by a solid substrate. Instead, more like a plasma membrane, it is mainly influenced by the properties of the underlying polymer and the nature/distribution of polymer-bilayer attachments. Polymer gel-supported lipid bilayers built using the capillary-assisted assembly approach show attractive self-healing properties, resulting in superior long-term stability relative to the SLB. We hypothesize that the described capillary-assisted assembly method can be applied to a wide range of polymeric materials and lipid compositions, opening exciting opportunities as an advanced model membrane system.

Translocation of Antimicrobial Peptides across Model Membranes: The Role of Peptide Chain Length.

Cushioned lipid bilayers are structures consisting of a lipid bilayer supported on a solid substrate with an intervening layer of soft material. They offer possibilities for studying the behavior and interactions of biological membranes more accurately under physiological conditions. In this work, we continue our studies of cushion formation induced by histatin 5 (24Hst5), focusing on the effect of the length of the peptide chain. 24Hst5 is a short, positively charged, intrinsically disordered saliva peptide, and here, both a shorter (14Hst5) and a longer (48Hst5) peptide variant were evaluated. Experimental surface active techniques were combined with coarse-grained Monte Carlo simulations to obtain information about these peptides. Results show that at 10 mM NaCl, both the shorter and the longer peptide variants behave like 24Hst5 and a cushion below the bilayer is formed. At 150 mM NaCl, however, no interaction is observed for 24Hst5. On the contrary, a cushion is formed both in the case of 14Hst5 and 48Hst5, and in the latter, an additional thick, diffuse, and highly hydrated layer of peptide and lipid molecules is formed, on top of the bilayer. Similar trends were observed from the simulations, which allowed us to hypothesize that positively charged patches of the amino acids lysine and arginine in all three peptides are essential for them to interact with and translocate over the bilayer. We therefore hypothesize that electrostatic interactions are important for the interaction between the solid-supported lipid bilayers and the peptide depending on the linear charge density through the primary sequence and the positively charged patches in the sequence. The understanding of how, why, and when the cushion is formed opens up the possibility for this system to be used in the research and development of new drugs and pharmaceuticals.

Fluorescence imaging of lamellipodin-mediated biomolecular condensates on solid supported lipid bilayer membranes.

Biomolecular condensates play a major role in numerous cellular processes, including several that occur on the surface of lipid bilayer membranes. There is increasing evidence that cellular membrane trafficking phenomena, including the internalization of the plasma membrane through endocytosis, are mediated by multivalent protein-protein interactions that can lead to phase separation. We have recently found that proteins involved in the clathrin-independent endocytic pathway named Fast Endophilin Mediated Endocytosis can undergo liquid-liquid phase separation (LLPS) in solution and on lipid bilayer membranes. Here, the protein solution concentrations required for phase separation to be observed are significantly smaller compared to those required for phase separation in solution. LLPS is challenging to systematically characterize in cellular systems in general, and on biological membranes in particular. Model membrane approaches are more suitable for this purpose as they allow for precise control over the nature and amount of the components present in a mixture. Here we describe a method that enables the imaging of LLPS domain formation on solid supported lipid bilayers. These allow for facile imaging, provide long-term stability, and avoid clustering of vesicles and vesicle-attached features (such as buds and tethers) in the presence of multi-valent membrane interacting proteins.

Surface nanoroughness impacts the formation and stability of supported lipid bilayers

Solid supported lipid bilayers (SLBs) are excellent platforms for studying the biophysical properties of cell membranes, as well as versatile biomimetic films for biotechnology applications. Among the existing approaches used to form SLBs, vesicle fusion and rupture onto solid supports is the most commonly employed one owing to its straightforward procedure. SLBs are typically formed on atomically flat and very hydrophilic surfaces, overlooking the influence of roughness and topography on membrane formation and organization. As a matter of fact, lipid bilayers in vivo are corrugated at the nanoscale level, as a result of interactions with proteins, fibrils, and other components within the intracellular and extracellular environment. Fundamental studies of the effect of surface roughness on SLBs are scarce and restricted to few contributions, where nanoroughness has shown to affect lipid mobility by a 5-fold decrease and inhibit domain growth in phase-separated membranes. In this work, the impact of nanoroughness on the formation and stability of SLBs onto SiO2 surfaces with different degrees of vertical and lateral surface roughness is studied. Combining quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy with force spectroscopy (AFM-FS), it is shown that nanoroughness affects the formation of SLBs by increasing the activation energy of vesicle fusion, rupture and spreading, and weakens the stability and lateral organization of the formed SLBs.

Engineering phosphatidylinositol-4,5-bisphosphate model membranes enriched in endocytic cargo: A neutron reflectometry, AFM and QCM-D structural study

The combination of in vitro models of biological membranes based on solid-supported lipid bilayers (SLBs) and of surface sensitive techniques, such as neutron reflectometry (NR), atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D), is well suited to provide quantitative information about molecular level interactions and lipid spatial distributions. In this work, cellular plasma membranes have been mimicked by designing complex SLB, containing phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) lipids as well as incorporating synthetic lipo-peptides that simulate the cytoplasmic tails of transmembrane proteins. The QCM-D results revealed that the adsorption and fusion kinetics of PtdIns4,5P2 are highly dependent of Mg2+. Additionally, it was shown that increasing concentrations of PtdIns4,5P2 leads to the formation of SLBs with higher homogeneity. The presence of PtdIns4,5P2 clusters was visualized by AFM. NR provided important insights about the structural organization of the various components within the SLB, highlighting that the leaflet symmetry of these SLBs is broken by the presence of CD4-derived cargo peptides. Finally, we foresee our study to be a starting point for more sophisticated in vitro models of biological membranes with the incorporation of inositol phospholipids and synthetic endocytic motifs.

Laurdan Discerns Lipid Membrane Hydration and Cholesterol Content.

Studies of biological membrane heterogeneity particularly benefit from the use of the environment-sensitive fluorescent probe Laurdan, for which shifts in the emission, produced by any stimulus (e.g., fluidity variations), are ascribed to alterations in hydration near the fluorophore. Ironically, no direct measure of the influence of the membrane hydration level on Laurdan spectra has been available. To address this, we investigated the fluorescence spectrum of Laurdan embedded in solid-supported lipid bilayers as a function of hydration and compared it with the effect of cholesterol─a major membrane fluidity regulator. The effects are illusively similar, and hence the results obtained with this probe should be interpreted with caution. The dominant phenomenon governing the changes in the spectrum is the hindrance of the lipid internal dynamics. Furthermore, we unveiled the intriguing mechanism of dehydration-induced redistribution of cholesterol between domains in the phase-separated membrane, which reflects yet another regulatory function of cholesterol.

Characterization of phosphoinositide domains in asymmetric giant unilamellar vesicles.

Phosphoinositides (PIPs) have been shown to mediate a broad range of cellular events by attracting proteins to specific cellular sites at distinct time points. This spatiotemporal control of protein function is rooted in the rich chemical functionality of the phosphoinositide headgroup, which not only gives rise to selective interactions with proteins but also results in unique physicochemical properties. Several studies have been directed at detailing PIP properties in model symmetric lipid bilayers, however, biological membranes exhibit compositional asymmetry between bilayer leaflets. It is unclear to what extent PIP properties are altered in such an environment. In the last decade, methods have been introduced that enable the fabrication of asymmetric lipid vesicles. We have extended the scope of one of these methods – Ca2+ initiated hemifusion of a symmetric giant unilamellar vesicles (sGUV) with solid supported lipid bilayers (SLB) – to enable the fabrication of asymmetric giant unilamellar vesicles (aGUVs) that exhibit phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the outer leaflet. We found that using Ca2+ as the initiator for sGUV/SLB hemifusion leads to a large disparity of PI(4,5)P2 content in the formed aGUVs. We attribute this disparity to the formation of macroscopic Ca2+/PI(4,5)P2 domains, which leads to sGUVs interacting with SLB regions rich or poor in PI(4,5)P2. In contrast, when Mg2+ is used as initiator, no macroscopic domains form in the SLB and hence, the aGUVs show significantly higher compositional uniformity. We find that Mg2+ concentrations as low as 1 mM are sufficient to initiate the hemifusion process. Using this method to obtain aGUVs, we investigate phosphoinositide domain properties for varying lipid compositions in the leaflet opposite the PIP containing leaflet (raft vs. non-raft lipid mixtures) and we explore the impact of bivalent cations on the lateral morphology of the respective aGUVs.

Hydrodynamics vs. membrane drag determines the mobility of bioengineered proteins during flow.

Biological membranes and the lipid-anchored proteins embedded in them experience shear forces, particularly in an environment such as the cardiovascular system. For example, Glypican-1, a GPI-anchored protein involved in flow sensing by cells, moves in the flow and direction to create a concentration gradient. The mobility of proteins and hence such formed concentration gradient profile can be affected by several factors such as protein size, shape, the viscosity of the surrounding media, protein-membrane anchor, and the composition of the membrane itself. To decouple the factors contributing to protein mobility, we developed a method to measure the flow mobility of proteins on a model system composed of lipid anchored proteins on solid-supported lipid bilayers (SSLBs). We combine microfluidics and confocal microscopy to monitor the protein gradients formed under controlled shear stress and hence measure the mobility of the protein. The protein mobility is determined by the balance of two forces experienced by the protein: the viscous drag force exerted by the membrane in which proteins are embedded and the hydrodynamic shear force exerted by the flow. The hydrodynamic shear force experienced by the protein under given stress is altered by protein size and shape whereas the viscous drag force exerted by the membranes depends on membrane composition and protein-membrane anchor. Here we independently vary these forces by creating a series of monomeric proteins and lipid mixtures. We successfully purified single and double-GFP-tagged monomeric Streptavidin protein and measured their mobilities in membranes with different lipid compositions. We obtain the parameters we can use to predict flow responses of protein in general.

QCM‐D Study of the Formation of Solid‐Supported Artificial Lipid Membranes: State‐of‐the‐Art, Recent Advances, and Perspectives

Solid‐supported lipid bilayers (SLBs) are excellent platforms for studying the biophysical properties of cell membranes. Among the existing approaches used to form SLBs, vesicle fusion and rupture onto solid supports are most commonly employed owing to their straightforward procedure. The current understanding of the mechanisms behind this approach has greatly benefited from the use of surface‐sensitive techniques, especially quartz crystal microbalance with dissipation monitoring (QCM‐D) in combination with other analytical techniques, such as atomic force microscopy (AFM) or localized surface plasmon resonance (LSPR). Herein, an overview of the pathways of vesicle adsorption and rupture under various experimental conditions is provided. Examples including recent findings of how the variation of the properties of lipid vesicles (size, charge), aqueous buffer (pH, ionic strength, osmotic pressure), and solid support (surface energy) affect the pathway mechanism of adsorption and rupture are provided. Recent reports on poorly understood properties such as surface roughness and topography are provided, together with the need for further studies relevant to biomimetic and sensing purposes.

Structural and mechanistic analysis of a tripartite ATP-independent periplasmic TRAP transporter

Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.

Monitoring the Progression of Cell-Free Expression of Microbial Rhodopsins by Surface Enhanced IR Spectroscopy: Resolving a Branch Point for Successful/Unsuccessful Folding.

The translocon-unassisted folding process of transmembrane domains of the microbial rhodopsins sensory rhodopsin I (HsSRI) and II (HsSRII), channelrhodopsin II (CrChR2), and bacteriorhodopsin (HsBR) during cell-free expression has been investigated by Surface-Enhanced Infrared Absorption Spectroscopy (SEIRAS). Up to now, only a limited number of rhodopsins have been expressed and folded into the functional holoprotein in cell free expression systems, while other microbial rhodopsins fail to properly bind the chromophore all-trans retinal as indicated by the missing visible absorption. SEIRAS experiments suggest that all investigated rhodopsins lead to the production of polypeptides, which are co-translationally inserted into a solid-supported lipid bilayer during the first hour after the in-vitro expression is initiated. Secondary structure analysis of the IR spectra revealed that the polypeptides form a comparable amount of α-helical structure during the initial phase of insertion into the lipid bilayer. As the process progressed (>1 h), only HsBR exhibited a further increase and association of α-helices to form a compact tertiary structure, while the helical contents of the other rhodopsins stagnated. This result suggests that the molecular reason for the unsuccessful cell-free expression of the two sensory rhodopsins and of CrChR2 is not due to the translation process, but rather to the folding process during the post-translational period. Taking our previous observation into account that HsBR fails to form a tertiary structure in the absence of its retinal, we infer that the chromophore retinal is an integral component of the compaction of the polypeptide into its tertiary structure and the formation of a fully functional protein.

Redox-Induced Lipid Vesicle Fusion Onto Electroactive Self-Assembled Monolayers

Supported lipid bilayers (SLBs) are popular model systems to study cell membrane functionalities and various biomolecular interaction forces. A key advantage of SLBs on metallic surfaces, is the application of spectroscopic, electrochemical, and surface plasmon resonance techniques for biomolecular recognition studies. The most common method for forming SLBs is vesicle fusion, which involves the adsorption, deformation, and rupture of small unilamellar vesicles (SUVs) of lipids from aqueous suspension to the substrate surface.1 The formation of continuous single bilayers by vesicle fusion can be problematic due to the many experimental parameters influencing vesicle rupture and bilayer spreading (i.e., lipid and vesicle properties, physicochemical characteristics of the surface, temperature, and solvent environment)1. Vesicle fusion typically works only with smooth hydrophilic surfaces, such as glass, silica, and mica. Although approaches have been developed to form bilayers on technologically relevant surfaces such gold (e.g., surface functionalization with hydrophilic organic films,2 solvent-assisted lipid bilayer formation3 or the addition of a vesicle-destabilizing agent4), there remains a need for active strategies that are fast, versatile, and scalable.We present a redox-induced approach for the formation of single bilayers on gold functionalized with electroactive self-assembled monolayers (SAMs) of ferrocenylalkanethiolates. The electrochemical oxidation of the SAM-bound ferrocene (Fc) to ferrocenium (Fc+) involves coupled electron transfer and ion pairing reactions. Counteranions from solution pair with the ferroceniums to stabilize the oxidized cations and neutralize the excess positive charge at the SAM/aqueous interface. The surface-confined redox reaction triggers the formation of single bilayer membranes from SUVs of anionic or zwitterionic phospholipids onto gold surfaces modified with the electroactive SAM. The ion pairing association of the charged lipid head groups with the electrogenerated ferroceniums drives the assembly of the phospholipids on the SAM surface to produce solid-supported bilayers of high surface coverage (≳ 90%) from gel- or fluid-phase forming phospholipids within minutes at room temperature. The redox-mediated strategy reported in this work is a conceptual advance in the preparation of solid-supported lipid bilayers. It is fast, insensitive to the phase state of the phospholipid in the vesicle precursor, and not limited to hydrophilic surfaces. References Richter, R. P.; Bérat, R.; Brisson, A. R., Formation of Solid-Supported Lipid Bilayers: An Integrated View. Langmuir 2006, 22 (8), 3497-3505.Silin, V. I.; Wider, H.; Woodward, J. T.; Valincius, G.; Offenhausser, A.; Plant, A. L., The Role of Surface Energy on the Formation of Hybrid Bilayer Membranes. J. Am. Chem. Soc. 2002, 124, 14676-14683.Ferhan, A. R.; Yoon, B. K.; Park, S.; Sut, T. N.; Chin, H.; Park, J. H.; Jackman, J. A.; Cho, N.-J., Solvent-Assisted Preparation of Supported Lipid Bilayers. Nat. Protoc. 2019, 14 (7), 2091-2118.Cho, N.-J.; Cho, S.-J.; Cheong, K. H.; Glenn, J. S.; Frank, C. W., Employing an Amphipathic Viral Peptide to Create a Lipid Bilayer on Au and TiO2. J. Am. Chem. Soc. 2007, 129 (33), 10050-10051. Figure 1

PH-induced insertion of pHLIP into a lipid bilayer: In-situ SEIRAS characterization of a folding intermediate at neutral pH

The pH low insertion peptide (pHLIP) is a pH-sensitive cell penetrating peptide that transforms from an unstructured coil on the membrane surface at pH > 7, to a transmembrane (TM) α-helix at pH < 5. By exploiting this unique property, pHLIP attracts interest as a potential tool for drug delivery and visualisation of acidic tissues produced by various maladies such as cancer, inflammation, hypoxia etc. Even though the structures of initial and end states of pHLIP insertion have been widely accepted, the intermediate structures in between these two states are less clear. Here, we have applied in situ Surface-Enhanced Infrared Absorption spectroscopy to examine the pH-induced insertion and folding processes of pHLIP into a solid-supported lipid bilayer. We show that formation of partially helical structure already takes place at pH only slightly below 7.0, but with the helical axis parallel to the membrane surface. The peptide starts to reorientate its helix from horizontal to vertical direction, accompanied by the insertion into the TM region at pH < 6.2. Further insertion into the TM region of the peptide results in an increase of inherent α-helical structure and complete secondary structure formation at pH 5.3. Analysis of the changes of the carboxylate vibrational bands upon pH titration shows two distinctive groups of aspartates and glutamates with pKa values of 4.5 and 6.3, respectively. Comparison to the amide bands of the peptide backbone suggests that the latter Asp/Glu groups are directly involved in the conformational changes of pHLIP in the respective intermediate states.

Solid-Supported Assembly Composed of n-Octyl-β-D-glucopyranoside and 1,2-Dioleoyl-sn-glycero-3-phosphocholine in Equilibrium with Its Ambient Aqueous Solution System Including Dispersed Assembly.

In the last few decades, the preparation of solid-supported lipid bilayers by immersing a solid substrate in an aqueous solution where the lipid is dissolved with the aid of a surfactant, followed by dilution of the solution, has been reported. In this study, we attempted to interpret the evolution of supported surfactant/lipid assemblies towards the supported lipid bilayer in terms of a phase equilibrium between the supported assembly phase and its ambient solution system consisting of the dispersed surfactant/lipid assembly phase and the bulk solution phase comprising monomeric surfactant and lipid. We characterized the supported assembly formed on hydrophilized Ge or mica substrates in equilibrium with aqueous solutions containing various concentrations of the nonionic surfactant, n-octyl-β-D-glucopyranoside (OG) and the amphoteric phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), using interaction-force-profile measurements by atomic force microscopy (AFM), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). We also investigated the ambient solution system using equilibrium dialysis to obtain the partition equilibrium profile of OG between the bulk solution and dispersed assembly phases in the micellar or vesicular states. These studies indicate that the properties of the supported assembly depend on the composition of the dispersed assembly and concentration of monomerically dissolved OG. Further, a type of micellar-bilayer state transition occurs in the supported assembly, roughly synchronized with that in the dispersed assembly.

Interaction of Caffeine with Model Lipid Membranes.

Caffeine is not only a widely consumed active stimulant, but it is also a model molecule commonly used in pharmaceutical sciences. In this work, by performing quartz–crystal microbalance and neutron reflectometry experiments we investigate the interaction of caffeine molecules with a model lipid membrane. We determined that caffeine molecules are not able to spontaneously partition from an aqueous environment, enriched in caffeine, into a bilayer. Caffeine could be however included in solid-supported lipid bilayers if present with lipids during self-assembly. In this case, thanks to surface-sensitive techniques, we determined that caffeine molecules are preferentially located in the hydrophobic region of the membrane. These results are highly relevant for the development of new drug delivery vectors, as well as for a deeper understanding of the membrane permeation role of purine molecules.

Real-time imaging of structure and dynamics of transmembrane biomolecules by FRET-induced single-molecule fluorescence attenuation.

Tracking the transmembrane topology and conformational dynamics of membrane proteins is key to understand their functions. It is however challenging to monitor position changes of individual proteins in cell membranes with high sensitivity and high resolution. We review on three single-molecule fluorescence imaging methods - SIFA, LipoFRET and QueenFRET - recently developed in our lab for studying the dynamics of membrane proteins. They can be applied, progressively, to investigate membrane proteins in solid-supported lipid bilayers, artificial liposome membranes and live-cell plasma membranes. The techniques take advantage of the energy transfer from a fluorophore to a cloud of quenchers and are able to extract in real time positions and position changes of a single fluorophore-labeled protein in the direction normal to the membrane surface. The methods have sub-nanometer precision and have proved powerful to investigate biomolecules interacting with bio-membranes.

From spherical compartments to polymer films: exploiting vesicle fusion to generate solid supported thin polymer membranes.

Solid supported polymer membranes as scaffold for the insertion of functional biomolecules provide the basis for mimicking natural membranes. They also provide the means for unraveling biomolecule-membrane interactions and engineering platforms for biosensing. Vesicle fusion is an established procedure to obtain solid supported lipid bilayers but the more robust polymer vesicles tend to resist fusion and planar membranes rarely form. Here, we build on vesicle fusion to develop a refined and efficient way to produce solid supported membranes based on poly(dimethylsiloxane)-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA) amphiphilic triblock copolymers. We first create thiol-bearing polymer vesicles (polymersomes) and anchor them on a gold substrate. An osmotic shock then provokes polymersome rupture and drives planar film formation. Prerequisite for a uniform amphiphilic planar membrane is the proper combination of immobilized polymersomes and osmotic shock conditions. Thus, we explored the impact of the hydrophobic PDMS block length of the polymersome on the formation and the characteristics of the resulting solid supported polymer assemblies by quarz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM) and spectroscopic ellipsometry (SE). When the PDMS block is short enough, attached polymersomes restructure in response to osmotic shock, resulting in a uniform planar membrane. Our approach to rapidly form planar polymer membranes by vesicle fusion brings many advantages to the development of synthetic planar membranes for bio-sensing and biotechnological applications.

A combination of electrochemistry and mass spectrometry to monitor the interaction of reactive species with supported lipid bilayers

Reactive oxygen and nitrogen species (RONS), e.g. generated by cold physical plasma (CPP) or photodynamic therapy, interfere with redox signaling pathways of mammalian cells, inducing downstream consequences spanning from migratory impairment to apoptotic cell death. However, the more austere impact of RONS on cancer cells remains yet to be clarified. In the present study, a combination of electrochemistry and high-resolution mass spectrometry was developed to investigate the resilience of solid-supported lipid bilayers towards plasma-derived reactive species in dependence of their composition. A 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer was undisturbed by 200 µM H2O2 (control) but showed full permeability after CPP treatment and space-occupying oxidation products such as PoxnoPC, PAzePC, and POPC hydroperoxide were found. Electron paramagnetic resonance spectroscopy demonstrated the presence of hydroxyl radicals and superoxide anion/hydroperoxyl radicals during the treatment. In contrast, small amounts of the intramembrane antioxidant coenzyme Q10 protected the bilayer to 50% and LysoPC was the only POPC derivative found, confirming the membrane protective effect of Q10. Such, the lipid membrane composition including the presence of antioxidants determines the impact of pro-oxidant signals. Given the differences in membrane composition of cancer and healthy cells, this supports the application of cold physical plasma for cancer treatment. In addition, the developed model using the combination of electrochemistry and mass spectrometry could be a promising method to study the effect of reactive species or mixes thereof generated by chemical or physical sources.

Channel-Water Dynamics are Correlated with the Conformational Changes of the M2 Viroporin Protein from Influenza a Virus

Viroporins are a class of viral ion channels that are involved in diverse processes entry, assembly and virulence. In the pursuit of understanding the mechanism of viroporins, knowledge of both structural and dynamic characteristics is essential. An experimental technique, sensitive to both structure and dynamics of the viroporin, is ideal for serving the purpose. Here, using the structure-sensitive two-dimensional infrared (2D IR) spectroscopy, with ultrafast time resolution, we have shown a direct correlation between channel-water dynamics and the conformational changes of the M2 viroporin from influenza A virus inserted into a solid-supported lipid bilayer. Our results show that a helix kink at higher pH (7.5) occludes the passage of water molecules into the channel, making it inactive. The stretched M2 structure at low pH (5.5) shows ultrafast hydrogen-bond dynamics between the backbone amide carbonyls and the water molecules, confirming the presence of water inside the channel in its active state. These results provide direct experimental evidence for a transporter-likeconduction mechanism, relating the channel-water dynamics with domain-specific structural changes in the viroporin. The results indicate that such experiments can be exploited for other topical coronaviruses (CoVs) viroporins, warranting investigation.