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Nanoparticle interactions with cellular membranes are controlled by molecular recognition reactions and regulate a multitude of biological processes, including virus infections, biological nanoparticle-mediated cellular communication, and drug delivery applications. Aided by the design of various supported cell membrane mimics, multiple methods have been employed to investigate these types of interactions, revealing information on nanoparticle coverage, interaction kinetics, as well as binding strength; however, precise quantification of the separation distance across which these delicate interactions occur remains elusive. Here, we demonstrate that carefully designed neutron reflectometry (NR) experiments followed by an attentive selection and application of suitable theoretical models offer a means to quantify the distance separating biological nanoparticles from a supported lipid bilayer (SLB) with sub-nanometer precision. The distance between the nanoparticles and SLBs was tuned by exploiting either direct adsorption or specific binding using DNA tethers with different conformations, revealing separation distances of around 1, 3, and 7 nm with nanometric accuracy. We also show that NR provides precise information on nanoparticle coverage, size distribution, material composition, and potential structural changes in the underlying planar SLB induced upon nanoparticle binding. The precision with which these parameters could be quantified should pave an attractive path for investigations of the interactions between nanoparticles and interfaces at length scales and resolutions that were previously inaccessible. This thus makes it possible to, for example, gain an in-depth understanding of the molecular recognition reactions of inorganic and biological nanoparticles with cellular membranes., Journal of the American Chemical Society, 144 (45), ISSN:0002-7863, ISSN:1520-5126

In this article, it is shown that by exposing commonly used lipids for biomembrane mimicking studies, to a solution containing the histidine-rich intrinsically disordered protein histatin 5, a protein cushion spontaneously forms underneath the bilayer. The underlying mechanism is attributed to have an electrostatic origin, and it is hypothesized that the observed behavior is due to proton charge fluctuations promoting attractive electrostatic interactions between the positively charged proteins and the anionic surfaces, with concomitant counterion release. Hence, we anticipate that this novel "green" approach of forming cushioned bilayers can be an important tool to mimic the cell membrane without the disturbance of the solid substrate, thereby achieving a further understanding of protein-cell interactions.

We measured the passive lipid flip-flop of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in solid supported lipid bilayers across their main gel to fluid (Lβ → Lα) phase transition. By performing time and temperature resolved neutron reflectometry experiments, we demonstrated that asymmetric systems prepared in the gel phase are stable for at least 24 hours. Lipid flip-flop was found to be intrinsically linked to the amount of lipid molecules in the fluid phase. Moreover, the increase of this amount during the broad phase transition was found to be the main key factor for the timing of the flip-flop process. By measuring different temperature scan rate, we could demonstrate that, in the case of supported bilayers and for the temperature investigated, the lipid flip flop is characterised by an activation energy of 50 kJ mol-1 and a timescale on the order of few hours. Our results demonstrate the origin on the discrepancies between passive flip-flop in bulk systems and at interfaces.

We studied the transleaflet coupling of compositionally asymmetric liposomes in the fluid phase. The vesicles were produced by cyclodextrin-mediated lipid exchange and contained dipalmitoyl phosphatidylcholine (DPPC) in the inner leaflet and different mixed-chain phosphatidylcholines (PCs) as well as milk sphingomyelin (MSM) in the outer leaflet. In order to jointly analyze the obtained small-angle neutron and X-ray scattering data, we adapted existing models of trans-bilayer structures to measure the overlap of the hydrocarbon chain termini by exploiting the contrast of the terminal methyl ends in X-ray scattering. In all studied systems, the bilayer-asymmetry has large effects on the lipid packing density. Fully saturated mixed-chain PCs interdigitate into the DPPC-containing leaflet and evoke disorder in one or both leaflets. The long saturated acyl chains of MSM penetrate even deeper into the opposing leaflet, which in turn has an ordering effect on the whole bilayer. These results are qualitatively understood in terms of a balance of entropic repulsion of fluctuating hydrocarbon chain termini and van der Waals forces, which is modulated by the interdigitation depth. Monounsaturated PCs in the outer leaflet also induce disorder in DPPC despite vestigial or even absent interdigitation. Instead, the transleaflet coupling appears to emerge here from a matching of the inner leaflet lipids to the larger lateral lipid area of the outer leaflet lipids. Graphical abstract

This review focuses on time-resolved neutron scattering, particularly time-resolved small angle neutron scattering (TR-SANS), as a powerful in situ noninvasive technique to investigate intra- and intermembrane transport and distribution of lipids and sterols in lipid membranes. In contrast to using molecular analogues with potentially large chemical tags that can significantly alter transport properties, small angle neutron scattering relies on the relative amounts of the two most abundant isotope forms of hydrogen: protium and deuterium to detect complex membrane architectures and transport processes unambiguously. This review discusses advances in our understanding of the mechanisms that sustain lipid asymmetry in membranes-a key feature of the plasma membrane of cells-as well as the transport of lipids between membranes, which is an essential metabolic process.

International audience; The proton pump transmembrane protein bacteriorhodopsin was successfully incorporated into planar floating lipid bilayers in gel and fluid phases, by applying a detergent-mediated incorporation method. The method was optimized on single supported bilayers by using quartz crystal microbalance, atomic force and fluorescence microscopy techniques. Neutron and X-ray reflectometry were used on both single and floating bilayers with the aim of determining the structure and composition of this membrane-protein system before and after protein reconstitution at sub-nanometer resolution. Lipid bilayer integrity and protein activity were preserved upon the reconstitution process. Reversible structural modifications of the membrane, induced by the bacteriorhodopsin functional activity triggered by visible light, were observed and characterized at the nanoscale.

HypothesesUnderstanding the mechanisms underlying lipolysis is crucial to address the ongoing obesity crisis and associated cardiometabolic disorders. Bile salts (BS), biosurfactants present in the small intestine, play key roles in lipid digestion and absorption. It is hypothesised that their contrasting functionalities – adsorption at oil/water interfaces and shuttling of lipolysis products away from these interfaces – are linked to their structural diversity. We investigate the interfacial films formed by two BS, sodium taurocholate (NaTC) and sodium taurodeoxycholate (NaTDC), differing by the presence or absence of a hydroxyl group on their steroid skeleton.ExperimentsTheir adsorption behaviour at the air/water interface and interaction with a phospholipid monolayer – used to mimic a fat droplet interface – were assessed by surface pressure measurements and ellipsometry, while interfacial morphologies were characterised in the lateral and perpendicular directions by Brewster angle microscopy, X-ray and neutron reflectometry, and molecular dynamics simulations.FindingsOur results provide a comprehensive molecular-level understanding of the mechanisms governing BS interfacial behaviour. NaTC shows a higher affinity for the air/water and lipid/water interfaces, and may therefore favour enzyme adsorption, whereas NaTDC exhibits a higher propensity for desorption from these interfaces, and may thus more effectively displace hydrolysis products from the interface, through dynamic exchange.

The outer membrane of Gram-negative bacteria is of great scientific interest because it mediates the action of antimicrobial agents. The membrane surface is composed of lipopolysaccharide (LPS) molecules with negatively charged oligosaccharide headgroups. To a certain fraction, LPSs additionally display linear polysaccharides termed O-side chains (OSCs). Structural studies on bacterial outer surfaces models, based on LPS monolayers at air-water interfaces, have so far dealt only with rough mutant LPSs lacking these OSCs. Here, we characterize monolayers of wild-type LPS from Escherichia coli O55:B5 featuring strain-specific OSCs in the presence of defined concentrations of monovalent and divalent ions. Pressure-area isotherms yield insight into in-plane molecular interactions and monolayer elastic moduli. Structural investigations by x-ray and neutron reflectometry reveal the saccharide conformation and allow quantifying the area per molecule and the fraction of LPS molecules carrying OSCs. The OSC conformation is satisfactorily described by the self-consistent field theory for end-grafted polymer brushes. The monolayers exhibit a significant structural response to divalent cations, which goes beyond generic electrostatic screening.

The use of glyceryl monooleate (GMO)-based nanoparticles has not yet been explored in overcoming the low bioavailability of Epigallocatechin-3-gallate (EGCG), a green tea polyphenol with a known anticancer activity. Since the inclusion of a guest molecule can affect the curvature and the supramolecular structure of fully hydrated GMO-based phase, the phase behavior of bulk and dispersed liquid crystalline systems containing EGCG were explored by Small Angle Neutron Scattering and X-Ray Diffraction experiments. Molecular Dynamic Simulations showed how the interaction of EGCG with polar heads of GMO strongly affects the curvature and packing of GMO phase. The EGCG encapsulation efficiency was determined in the nanodispersions and their size studied by Dynamic Light Scattering and Atomic Force Microscopy. A nanodispersed formulation has been optimized with a cytotoxic effect more than additive of GMO and EGCG.

Hypotheses\ud \ud Bile salts (BS) are biosurfactants released into the small intestine, which play key and contrasting roles in lipid digestion: they adsorb at interfaces and promote the adsorption of digestive enzymes onto fat droplets, while they also remove lipolysis products from that interface, solubilising them into mixed micelles. Small architectural variations on their chemical structure, specifically their bile acid moiety, are hypothesised to underlie these conflicting functionalities, which should be reflected in different aggregation and solubilisation behaviour.\ud \ud \ud \ud Experiments\ud \ud The micellisation of two BS, sodium taurocholate (NaTC) and sodium taurodeoxycholate (NaTDC), which differ by one hydroxyl group on the bile acid moiety, was assessed by pyrene fluorescence spectroscopy, and the morphology of aggregates formed in the absence and presence of fatty acids (FA) and monoacylglycerols (MAG) – typical lipolysis products – was resolved by small-angle X-ray/neutron scattering (SAXS, SANS) and molecular dynamics simulations. The solubilisation by BS of triacylglycerol-incorporating liposomes – mimicking ingested lipids – was studied by neutron reflectometry and SANS.\ud \ud \ud \ud Findings\ud \ud Our results demonstrate that BS micelles exhibit an ellipsoidal shape. NaTDC displays a lower critical micellar concentration and forms larger and more spherical aggregates than NaTC. Similar observations were made for BS micelles mixed with FA and MAG. Structural studies with liposomes show that the addition of BS induces their solubilisation into mixed micelles, with NaTDC displaying a higher solubilising capacity.

The maturation of coronavirus SARS-CoV-2, which is the etiological agent at the origin of the COVID-19 pandemic, requires a main protease Mpro to cleave the virus-encoded polyproteins. Despite a wealth of experimental information already available, there is wide disagreement about the Mpro monomer-dimer equilibrium dissociation constant. Since the functional unit of Mpro is a homodimer, the detailed knowledge of the thermodynamics of this equilibrium is a key piece of information for possible therapeutic intervention, with small molecules interfering with dimerization being potential broad-spectrum antiviral drug leads. In the present study, we exploit small angle x-ray scattering (SAXS) to investigate the structural features of the SARS-CoV-2 Mpro monomer-dimer equilibrium, by revealing the corresponding equilibrium dissociation constant and the associated thermodynamic parameters. SAXS is also used to study how the Mpro dissociation process is affected by small inhibitors selected through combinatorial design. Our results show that a clear picture connecting the ability of inhibitors to disrupt the Mpro dimerization with the loss of catalytic activity cannot be provided, thus highlighting the possible role of allosteric effects for the regulation of Mpro functionality.

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.

This review focuses on time-resolved neutron scattering, particularly time-resolved small angle neutron scattering (TR-SANS), as a powerful in situ noninvasive technique to investigate intra- and intermembrane transport and distribution of lipids and sterols in lipid membranes. In contrast to using molecular analogues with potentially large chemical tags that can significantly alter transport properties, small angle neutron scattering relies on the relative amounts of the two most abundant isotope forms of hydrogen: protium and deuterium to detect complex membrane architectures and transport processes unambiguously. This review discusses advances in our understanding of the mechanisms that sustain lipid asymmetry in membranes-a key feature of the plasma membrane of cells-as well as the transport of lipids between membranes, which is an essential metabolic process.

The maturation of coronavirus SARS-CoV-2, which is the etiological agent at the origin of the COVID-19 pandemic, requires a main protease Mpro to cleave the virus-encoded polyproteins. Despite a wealth of experimental information already available, there is wide disagreement about the Mpro monomer-dimer equilibrium dissociation constant. Since the functional unit of Mpro is a homodimer, the detailed knowledge of the thermodynamics of this equilibrium is a key piece of information for possible therapeutic intervention, with small molecules interfering with dimerization being potential broad-spectrum antiviral drug leads. In the present study, we exploit Small Angle X-ray Scattering (SAXS) to investigate the structural features of SARS-CoV-2 Mpro in solution as a function of protein concentration and temperature. A detailed thermodynamic picture of the monomer-dimer equilibrium is derived, together with the temperature-dependent value of the dissociation constant. SAXS is also used to study how the Mpro dissociation process is affected by small inhibitors selected by virtual screening. We find that these inhibitors affect dimerization and enzymatic activity to a different extent and sometimes in an opposite way, likely due to the different molecular mechanisms underlying the two processes. The Mpro residues that emerge as key to optimize both dissociation and enzymatic activity inhibition are discussed.

International audience; Methylcellulose (MC) has a demonstrated capacity to reduce fat absorption, hypothetically through bile salt (BS) activity inhibition. We investigated MC cholesterol-lowering mechanism, and compared the influence of two BS, sodium taurocholate (NaTC) and sodium taurodeoxycholate (NaTDC), which differ slightly by their architecture and exhibit contrasting functions during lipolysis. BS/MC bulk interactions were investigated by rheology, and BS behaviour at the MC/water interface studied with surface pressure and ellipsometry measurements. In vitro lipolysis studies were performed to evaluate the effect of BS on MC-stabilised emulsion droplets microstructure, with confocal microscopy, and free fatty acids release, with the pH-stat method. Our results demonstrate that BS structure dictates their interactions with MC, which, in turn, impact lipolysis. Compared to NaTC, NaTDC alters MC viscoelasticity more significantly, which may correlate with its weaker ability to promote lipolysis, and desorbs from the interface at lower concentrations, which may explain its higher propensity to destabilise emulsions.

Over the last 10 years, neutron reflectometry (NR) has emerged as a powerful technique for the investigation of biologically relevant thin films. The great advantage of NR with respect to many other surface-sensitive techniques is its sub-nanometer resolution that enables structural characterizations at the molecular level. In the case of bio-relevant samples, NR is non-destructive and can be used to probe thin films at buried interfaces or enclosed in bulky sample environment equipment. Moreover, recent advances in biomolecular deutera-tion enabled new labeling strategies to highlight certain structural features and to resolve with better accuracy the location of chemically similar molecules within a thin film. In this chapter I will describe some applications of NR to bio-relevant samples and discuss some of the data analysis approaches available for biological thin films. In particular, examples on the structural characterization of biomembranes, protein films and protein-lipid interactions will be described.

The adsorption of myoglobin (Mb) onto nanoporous thin films build up using block‐copolymers (BCPs) is analyzed. The nanoporous thin films, fabricated by exploiting self‐assembly of lamellar BCPs and the concept of sacrificial block, are characterized by a well‐defined morphology containing nanochannels of width ≈20 nm delimited by polystyrene (PS) domains, decorated with pendant poly(ethylene oxide) (PEO) chains. The adsorption of Mb onto the nanoporous material is studied by means of UV–visible spectroscopy, quartz crystal microbalance, and neutron reflectometry measurements. In order to determine the role of nanopores, experiments are also conducted by using supports made of nonporous PS thin films and nude glass slides. The results indicate that the BCP‐nanoporous material exhibits a remarkably higher adsorption capability than PS and glass. As PEO exhibits a low degree of protein adsorption, this result may be essentially attributed to the presence of the nanopores. The large surface area, the opened pore structure, and the trapping effect of the pores are the main factors determining the increased Mb adsorption capability of the BCP‐based support. Yet, the presence of PEO chains decorating the PS walls at porous surface does not prevent Mb biomolecules to establish good interactions with the support.

The outer membrane (OM) of Gram-negative bacteria is a complex and asymmetric bilayer that antimicrobial peptides must disrupt in order to provoke the cell lysis. The inner and external leaflets of the OM are mainly composed of phospholipids (PL), and lipopolysaccharide (LPS), respectively. Supported lipid bilayers are interesting model systems to mimic the lipid asymmetric scaffold of the OM and determine the quantitative and mechanistic effect of antimicrobial agents, using complementary physicochemical techniques. We report the formation of asymmetric PL/LPS bilayers using the Langmuir-Blodgett/Langmuir-Schaefer technique on two different surfaces (sapphire and mica) with synthetic phospholipids constituting the inner leaflet and bacteria-extracted mutant LPS making up the outer one. The combination of neutron reflectometry and atomic force microscopy techniques allowed the examination of the asymmetric scaffold structure along the normal to the interface and its surface morphology in buffer conditions. Our results allow discrimination of two structurally related peptides, one neutral and inactive, and the other cationic and active. The active cationic plasticin PTCDA1-KF disrupts the asymmetric OM at relevant concentrations through a carpeting scenario characterized by a dramatic removal of lipid molecules from the surface.

The safe application of nanotechnology devices in biomedicine requires fundamental understanding on how they interact with and affect the different components of biological systems. In this respect, the cellular membrane, the cell envelope, certainly represents an important target or barrier for nanosystems. Here we report on the interaction between functionalized SuperParamagnetic Iron Oxide Nanoparticles (SPIONs), promising contrast agents for Magnetic Resonance Imaging (MRI), and lipid bilayers that mimic the plasma membrane. Neutron Reflectometry, supported by Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) experiments, was used to characterize this interaction by varying both SPION coating and lipid bilayer composition. In particular, the interaction of two different SPIONs, functionalized with a cationic surfactant and a zwitterionic phospholipid, and lipid bilayers, containing different amount of cholesterol, were compared. The obtained results were further validated by Dynamic Light Scattering (DLS) measurements and Cryogenic Transmission Electron Microscopy (Cryo-TEM) images. None of the investigated functionalized SPIONs were found to disrupt the lipid membrane. However, in all case we observed the attachment of the functionalized SPIONs onto the surface of the bilayers, which was affected by the bilayer rigidity, i.e. the cholesterol concentration.

The naturally occurring peptide indolicidin from bovine neutrophils exhibits strong biological activity against a broad spectrum of microorganisms. This is believed to arise from selective interactions with the negatively charged cytoplasmic lipid membrane found in bacteria. We have investigated the peptide interaction with supported lipid model membranes using a combination of complementary surface sensitive techniques: neutron reflectometry (NR), atomic force microscopy (AFM), and quartz crystal microbalance with dissipation monitoring (QCM-D). The data are compared with small-angle X-ray scattering (SAXS) results obtained with lipid vesicle/peptide solutions. The peptide membrane interaction is shown to be significantly concentration dependent. At low concentrations, the peptide inserts at the outer leaflet in the interface between the headgroup and tail core. Insertion of the peptide results in a slight decrease in the lipid packing order of the bilayer, although not sufficient to cause membrane thinning. By increasing the indolicidin concentration well above the physiologically relevant conditions, a deeper penetration of the peptide into the bilayer and subsequent lipid removal take place, resulting in a slight membrane thinning. The results suggest that indolicidin induces lipid removal and that mixed indolicidin-lipid patches form on top of the supported lipid bilayers. Based on the work presented using model membranes, indolicidin seems to act through the interfacial activity model rather than through the formation of stable pores.

The dynamical and structural properties of caffeine solutions at the solubility limit have been investigated as a function of temperature by means of MD simulations, static and dynamic light scattering, and small angle neutron scattering experiments. A clear picture unambiguously supported by both experiment and simulation emerges: caffeine self-aggregation promotes the formation of two distinct types of clusters: linear aggregates of stacked molecules, formed by 2-14 caffeine molecules depending on the thermodynamic conditions and disordered branched aggregates with a size in the range 1000-3000 Å. While the first type of association is well-known to occur under room temperature conditions for both caffeine and other purine systems, such as nucleotides, the presence of the supramolecular aggregates has not been reported previously. MD simulations indicate that branched structures are formed by caffeine molecules in a T-shaped arrangement. An increase of the solubility limit (higher temperature but also higher concentration) broadens the distribution of cluster sizes, promoting the formation of stacked aggregates composed by a larger number of caffeine molecules. Surprisingly, the effect on the branched aggregates is rather limited. Their internal structure and size do not change considerably in the range of solubility limits investigated.

Auroreis a free software application based on MATLAB scripts designed for the graphical analysis, inspection and simulation of neutron reflectivity data. Its architecture, combined with graphics and other advantages of the MATLAB environment, should allow continued development of this software and inclusion of new features and analysis methods. The development of the software was driven by the necessity for a non-commercial open-source application for the analysis of neutron reflectivity data.Auroreprovides a robust and reliable method for evaluation of parameter uncertainty, a feature almost absent in similar software applications. In the present paper the main functionalities of the software are presented, together with a comprehensive description of the modeling approaches available at the moment. The code is released under a Creative Commons Attribution Non-Commercial License V2.0. The software application can be downloaded at http://aurorenr.sourceforge.net/.

Over the last two decades, supported lipid bilayers (SLBs) have been extensively used as model systems to study cell membrane structure and function. While SLBs have been traditionally produced from simple lipid mixtures, there has been a recent surge in compositional complexity to better mimic cellular membranes and thereby bridge the gap between classic biophysical approaches and cell experiments. To this end, native cellular membrane derived SLBs (nSLBs) have emerged as a new category of SLBs. As a new type of biomimetic material, an analytical workflow must be designed to characterize its molecular composition and structure. Herein, we demonstrate how a combination of fluorescence microscopy, neutron reflectometry, and secondary ion mass spectrometry offers new insights on structure, composition, and quality of nSLB systems formed using so-called hybrid vesicles, which are a mixture of native membrane material and synthetic lipids. With this approach, we demonstrate that the nSLB formed a continuous structure with complete mixing of the synthetic and native membrane components and a molecular stoichiometry that essentially mirrors that of the hybrid vesicles. Furthermore, structural investigation of the nSLB revealed that PEGylated lipids do not significantly thicken the hydration layer between the bilayer and substrate when on silicon substrates; however, nSLBs do have more topology than their simpler, purely synthetic counterparts. Beyond new insights regarding the structure and composition of nSLB systems, this work also serves to guide future researchers in producing and characterizing nSLBs from their cellular membrane of choice.

Fabrication of synthetic surfaces that reproduce structure and function of biological membranes is an open challenge. This work reports the first example of supported lipid bilayers obtained from extracellular vesicles (EVSLBs). EVSLBs harness and pattern in two dimensions key properties of extracellular vesicle (EV) membranes, which present intermediate complexity between synthetic mimics and natural membranes and innate link to phenotype and function of the originating cells. Silica-supported EVSLBs are formed from nanosized EVs—separated from culture media of prostate cancer model TRAMP-C2 murine cells—following a characteristic crowding-fusion pathway. They display peculiar properties at different length scales, such as 2.5 nm roughness, lipid-raft-like domains, and cushioned patches (with the cushion filled with the native EV biomolecular cargo), which preserve the native EV membrane orientation with the proto-oncogene tyrosine-protein kinase Src (c-Src) accessible to antibody recognition.

Interactions between soft interfaces govern the behavior of emulsions and foams and crucially influence the functions of biological entities like membranes. To understand the character of these interactions, detailed insight into the interfaces' structural response in terms of molecular arrangements and conformations is often essential. This requires the realization of controlled interaction conditions and surface-sensitive techniques capable of resolving the structure of buried interfaces. Here, we present a new approach to determine the distance-dependent structure of interacting soft interfaces by neutron reflectometry. A solid/water interface and a water/oil interface are functionalized independently and initially macroscopically separated. They are then brought into contact and structurally characterized under interacting conditions. The nanometric distance between the two interfaces can be varied via the exertion of osmotic pressures. Our first experiments on lipid-anchored polymer brushes interacting across water with solid-grafted polyelectrolyte brushes and with bare silicon surfaces reveal qualitatively different interaction scenarios depending on the chemical composition of the two involved interfaces.

We set up a facile approach for fabrication of supports with tailored nanoporosity for immobilization of enzymes. To this aim block copolymers (BCPs) self-assembly has been used to prepare nanostructured thin films with well-defined architecture containing pores of tailorable size delimited by walls with tailorable degree of hydrophilicity. In particular, we employed a mixture of polystyrene-block-poly(l-lactide) (PS-PLLA) and polystyrene-block-poly(ethylene oxide) (PS-PEO) diblock copolymers to generate thin films with a lamellar morphology consisting of PS lamellar domains alternating with mixed PEO/PLLA blocks lamellar domains. Selective basic hydrolysis of the PLLA blocks generates thin films, patterned with nanometric channels containing hydrophilic PEO chains pending from PS walls. The shape and size of the channels and the degree of hydrophilicity of the pores depend on the relative length of the blocks, the molecular mass of the BCPs, and the composition of the mixture. The strength of our approach is demonstrated in the case of physical adsorption of the hemoprotein peroxidase from horseradish (HRP) using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with H2O2 as substrate. The large surface area, the tailored pore sizes, and the functionalization with hydrophilic PEO blocks make the designed nanostructured materials suitable supports for the nanoconfinement of HRP biomolecules endowed with high catalytic performance, no mass-transfer limitations, and long-term stability.

International audience; Islet amyloid polypeptide (IAPP) is responsible for cell depletion in the pancreatic islets of Langherans, and for multiple pathological consequences encountered by patients suffering from type 2 Diabetes Mellitus. We have examined the amyloidogenicity and cytotoxic mechanisms of this peptide by investigating model-membrane permeation and structural effects of fragments of the human IAPP and several rat IAPP mutants. In vitro experiments and molecular dynamics simulations reveal distinct physical segregation, membrane permeation, and amyloid aggregation processes that are mediated by two separate regions of the peptide. These observations suggest a "detergent-like" mechanism, where lipids are extracted from the bilayer by the N-terminus of IAPP, and integrated into amyloid aggregates. The amyloidogenic aggregation would kinetically compete with the process of membrane permeation and, therefore, inhibit it. This hypothesis represents a new perspective on the mechanism underlying the membrane disruption by amyloid peptides, and could influence the development of new therapeutic strategies.

Inter- and intrabilayer transfer of phospholipid molecules was investigated by neutron reflectometry. The structure of solid supported lipid bilayers exposed to a solution of isotopically labeled vesicles was monitored as a function of temperature, time, and vesicle concentration. Lipid interbilayer exchange was shown to be the time limiting process, while lipid intrabilayer movement, the so-called flip-flop, was too fast to be visualized within the experimental acquisition time. The exchange process was characterized by an Arrhenius-like behavior and the activation energy of the process was concentration-independent. The results are discussed and compared extensively with the literature available on the topic.

Trichogin GA IV (GAIV) is an antimicrobial peptide of the peptaibol family, like the extensively studied alamethicin (Alm). GAIV acts by perturbing membrane permeability. Previous data have shown that pore formation is related to GAIV aggregation and insertion in the hydrophobic core of the membrane. This behavior is similar to that of Alm and in agreement with a barrel-stave mechanism, in which transmembrane oriented peptides aggregate to form a channel. However, while the 19-amino acid long Alm has a length comparable to the membrane thickness, GAIV comprises only 10 amino acids, and its helix is about half the normal bilayer thickness. Here, we report the results of neutron reflectivity measurements, showing that GAIV inserts in the hydrophobic region of the membrane, causing a significant thinning of the bilayer. Molecular dynamics simulations of GAIV/membrane systems were also performed. For these studies we developed a novel approach for constructing the initial configuration, by embedding the short peptide in the hydrophobic core of the bilayer. These calculations indicated that in the transmembrane orientation GAIV interacts strongly with the polar phospholipid headgroups, drawing them towards its N- and C-termini, inducing membrane thinning and becoming able to span the bilayer. Finally, vesicle leakage experiments demonstrated that GAIV activity is significantly higher with thinner membranes, becoming similar to that of Alm when the bilayer thickness is comparable to its size. Overall, these data indicate that a barrel-stave mechanism of pore formation might be possible for GAIV and for similarly short peptaibols despite their relatively small size.

Neutron scattering techniques have been employed to investigate 1,2-dimyristoyl-sn -glycero-3-phosphocholine (DMPC) membranes in the form of multilamellar vesicles (MLVs) and deposited, stacked multilamellar-bilayers (MLBs), covering transitions from the gel to the liquid phase. Neutron diffraction was used to characterise the samples in terms of transition temperatures, whereas elastic incoherent neutron scattering (EINS) demonstrates that the dynamics on the sub-macromolecular length-scale and pico- to nano-second time-scale are correlated with the structural transitions through a discontinuity in the observed elastic intensities and the derived mean square displacements. Molecular dynamics simulations have been performed in parallel focussing on the length-, time- and temperature-scales of the neutron experiments. They correctly reproduce the structural features of the main gel-liquid phase transition. Particular emphasis is placed on the dynamical amplitudes derived from experiment and simulations. Two methods are used to analyse the experimental data and mean square displacements. They agree within a factor of 2 irrespective of the probed time-scale, i.e. the instrument utilized. Mean square displacements computed from simulations show a comparable level of agreement with the experimental values, albeit, the best match with the two methods varies for the two instruments. Consequently, experiments and simulations together give a consistent picture of the structural and dynamical aspects of the main lipid transition and provide a basis for future, theoretical modelling of dynamics and phase behaviour in membranes. The need for more detailed analytical models is pointed out by the remaining variation of the dynamical amplitudes derived in two different ways from experiments on the one hand and simulations on the other.

The dynamics of hyaluronate-based hydrogels has been investigated by quasielastic neutron scattering (QENS). Hyaluronate (HYA) has been compared, in the same conditions of temperature and polymer concentration, to a chemically modified form, HYADD, in which the backbone has been grafted with a hexadecyl (C(16)) side-chain with a degree of substitution of about 2% (mol/mol). This modification increases the hydrophobicity of the polysaccharide and leads to a stable gel already at polymer concentration of 0.3% (w/v), yielding a viscosupplementation with less quantity of polysaccharide. The time-scale covered by our measurements probes both water and segmental biopolymer motions. In both systems, the local dynamics of the network in the ps time-scale is mostly due to local reorientational motions of side groups. Such motions are not significantly affected by the small amount of aliphatic chains forming the hydrophobic junctions in HYADD. The diffusivity of water in both HYA and HYADD coincides with that of pure water within the experimental uncertainty. This result confirms previous ones on the dynamics of water in HYA solutions and it is of relevance for biomedical applications of hyaluronate-based systems because it affects the diffusive processes of metabolites and their interaction with tissues.

Characterization of switchable microgels is a major task in drug delivery science. The study of soft polymeric devices requires a combined use of spectroscopy, microscopy, and scattering approaches enabling the characterization of nanostructured features across a volume phase transition. In this work the structural changes of poly(vinyl alcohol) based thermoreversible microgel particles which incorporate p(NiPAAm-co-methacrylate) chains across the transition temperature occurring at 33 °C have been addressed by utilizing reciprocal and direct space approaches such as small angle neutron scattering, SANS, dynamic light scattering, DLS, soft transmittance X-ray microscopy, STXM, and confocal laser scanning microscopy, CLSM, respectively. The comparison between the results obtained from those approaches allows an evaluation of the driving forces acting in the transition and reveals the changes in the microgel structure at nanoscale level. The structure of the poly(vinyl alcohol) based microgel particles, inc...

In recent biomedical studies different nanocarrier systems have been proposed for ´smart´ drug delivery. The engineering of these systems requires as a prerequisite a detailed knowledge of their structure and dynamics at the molecular level. Quasielastic neutron scattering is an ideal tool for dynamic studies of these complex systems since it provides information on molecular motions in a time window which is important to relate the local dynamics to the macroscopic functional properties of the drug vectors. Some selected examples referring to different nanostructures will be analysed in the following.

The effect of lipophilic additives (excipients and drugs) on the behavior of lipid/saccharide nanoparticles has been investigated by incoherent elastic neutron scattering. Temperature scans from 20 K to 350 K have been performed on lecithin/chitosan particles loaded with isopropyl myristate and cetyl-stearyl alcohol, two lipophilic molecules with different melting temperatures which are commonly added to improve drug loading efficiency. In a similar way the effect of tamoxifen citrate, a lipophilic drug frequently used in breast cancer therapy, has also been studied. The different melting points of the two excipients affect mostly the low-temperature behavior of the nanoparticles. At physiological temperatures they both improve the particle flexibility. On the other hand addition of tamoxifen leads to stiffer structures and to lower amounts of released drug. The macroscopic features of the drug release appear to be correlated to the microscopic flexibility determined by neutron scattering. The data confirm also the role of chitosan as a stiffening and stabilizing agent of the lipid particles. © 2010 The Royal Society of Chemistry.

Nano- and microparticles composed of saccharide and lipid systems are extensively investigated for applications as highly biocompatible drug carriers. A detailed understanding of particle–solvent interactions is of key importance in order to tailor their characteristics for delivering drugs with specific chemical properties. Here we report results of a quasielastic neutron scattering (QENS) investigation on lecithin/chitosan nanoparticles prepared by autoassembling the two components in an aqueous solution. The measurements were performed at room temperature on lyophilized and H 2 O hydrated nanoparticles ( h = 0.47 w H 2 O/w hydrated sample). In the latter, hydration water is mostly enclosed inside the nanoparticles; its dynamics is similar to that of bulk water but with a significant decrease in diffusivity. The scattering from the nanoparticles can be described by a simple model of confined diffusion. In the lyophilized state only hydrogens belonging to the polar heads are seen as mobile within the experimental time-window. In the hydrated sample the diffusive dynamics involves also a significant part of the hydrogens in the lipid tails.

POP-Ade (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyladenosine) is a biocompatible anionic nucleolipid with the DNA nucleoside, Adenosine, in the polar headgroup. We have studied the affinity of nucleic acids of different contour length, composition and structure toward supported lipid bilayers (SLB) composed of POP-Ade mixed with the zwitterionic phospholipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) using quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). In order to highlight the specificity of the nucleic acid interaction, the results were compared with data obtained for SLB containing the anionic phospholipid POPG (1-palmitoyl-2-oleoyl-sn-phosphatidyl-glycerol) replacing POP-Ade. Our results demonstrate that the presence of a nucleobase headgroup provides the bilayers with the ability to bind single stranded nucleic acids in a selective fashion, according to a Watson-Crick pattern. In addition the interaction with double stranded nucleic acids was strengthened. Overall, these findings represent fundamental information for the design of biocompatible DNA vectors with DNA-RNA-based amphiphiles.

Neutron scattering studies on mimetic biomembranes are currently limited by the low availability of deuterated unsaturated lipid species. In the present work, results from the first neutron diffraction experiments on fully deuterated lipid extracts from the yeastPichia pastorisare presented. The structural features of these fully deuterated lipid stacks are compared with those of their hydrogenous analogues and with other similar synthetic systems. The influence of temperature and humidity on the samples has been investigated by means of small momentum-transfer neutron diffraction. All of the lipid extracts investigated self-assemble into multi-lamellar stacks having different structural periodicities; the stacking distances are affected by temperature and humidity without altering the basic underlying arrangement. At high relative humidity the deuterated and hydrogenous samples are similar in their multi-lamellar arrangement, being characterized by two main periodicities of ∼75 and ∼110 Å reflecting the presence of a large number of polar phospholipid molecules. Larger differences are found at lower relative humidity, where hydrogenous lipids are characterized by a larger single lamellar structure than that observed in the deuterated samples. In both cases the heterogeneity in composition is reflected in a wide structural complexity. The different behaviour upon dehydration can be related to compositional differences in the molecular composition of the two samples, which is attributed to metabolic effects related to the use of perdeuterated growth media.

International audience; The myelin sheath is a tightly packed, multilayered membrane structure wrapped around selected nerve axons in the central and the peripheral nervous system. Because of its electrical insulation of the axons, which allows fast, saltatory nerve impulse conduction, myelin is crucial for the proper functioning of the vertebrate nervous system. A subset of myelin-specific proteins is well-defined, but their influence on membrane dynamics, i.e. myelin stability, has not yet been explored in detail. We investigated the structure and the dynamics of reconstituted myelin membranes on a pico- to nanosecond timescale, influenced by myelin basic protein (MBP) and myelin protein 2 (P2), using neutron diffraction and quasi-elastic neutron scattering. A model for the scattering function describing molecular lipid motions is suggested. Although dynamical properties are not affected significantly by MBP and P2 proteins, they act in a highly synergistic manner influencing the membrane structure.

Fixation is a key element for many clinical researches requiring long exposure time and resting of the tissue to avoid experimental artifacts; condition which is not easy achieved during in-vivo experiments. Several ex-vivo, e.g. post-mortem, fixation processes are commonly used, from chemical fixation to cryo-preserving methods, both having advantages and drawbacks and eventually affecting structural and dynamical properties of water within the tissue. In this paper we make use of neutron scattering technique to compare the dynamical properties of ex-vivo sections of cerebral bovine hemisphere preserved using formalin fixation and cryo-protection. Neutron scattering is a unique technique able to monitor water diffusion at atomic scale. Results suggest reduction of global water dynamics induced by the formalin fixation protocol at room temperature.