Boosting Membrane Interactions and Antimicrobial Effects of Photocatalytic Titanium Dioxide Nanoparticles by Peptide Coating.

Photocatalytic nanoparticles offer antimicrobial effects under illumination due to the formation of reactive oxygen species (ROS), capable of degrading bacterial membranes. ROS may, however, also degrade human cell membranes and trigger toxicity. Since antimicrobial peptides (AMPs) may display excellent selectivity between human cells and bacteria, these may offer opportunities to effectively "target" nanoparticles to bacterial membranes for increased selectivity. Investigating this, photocatalytic TiO2 nanoparticles (NPs) are coated with the AMP LL-37, and ROS generation is found by C11 -BODIPY to be essentially unaffected after AMP coating. Furthermore, peptide-coated TiO2 NPs retain their positive ζ-potential also after 1-2 h of UV illumination, showing peptide degradation to be sufficiently limited to allow peptide-mediated targeting. In line with this, quartz crystal microbalance measurements show peptide coating to promote membrane binding of TiO2 NPs, particularly so for bacteria-like anionic and cholesterol-void membranes. As a result, membrane degradation during illumination is strongly promoted for such membranes, but not so for mammalian-like membranes. The mechanisms of these effects are elucidated by neutron reflectometry. Analogously, LL-37 coating promoted membrane rupture by TiO2 NPs for Gram-negative and Gram-positive bacteria, but not for human monocytes. These findings demonstrate that AMP coating may selectively boost the antimicrobial effects of photocatalytic NPs.

Grafted chitosan thin films of various degrees of acetylation as a reusable platform for the investigation of biological interactions.

Surface treatment by adhesive polymers is a promising solution to immobilize and study bacteria cells through microscopic assays and, for example, control their growth or determine their susceptibility to antibiotic treatment. The stability of such functional films in wet conditions is crucial, as the film degradation would compromise a persistent use of the coated devices. In this work, low roughness chitosan thin films of degrees of acetylation (DA) ranging from 0.5 % to 49 % were chemically grafted onto silicon and glass substrates and we have demonstrated how the physicochemical properties of the surfaces and the bacterial response were DA-dependent. A fully deacetylated chitosan film presented an anhydrous crystalline structure while the hydrated crystalline allomorph was the preferred structure at higher DA. Moreover, their hydrophilicity increased at higher DA, leading to higher film swelling. Low DA chitosan-grafted substrate favored bacterial growth away from the surface and could be envisioned as bacteriostatic surfaces. Contrarily, an optimum of Escherichia coli adhesion was found for substrates modified with chitosan of DA = 35 %: these surfaces are adapted for the study of bacterial growth and antibiotic testing, with the possibility of reusing the substrates without affecting the grafted film - ideal for limiting single-use devices.

Competitive Adsorption of a Monoclonal Antibody and Nonionic Surfactant at the PDMS/Water Interface.

Interfacial adsorption of monoclonal antibodies (mAbs) can cause structural deformation and induce undesired aggregation and precipitation. Nonionic surfactants are often added to reduce interfacial adsorption of mAbs which may occur during manufacturing, storage, and/or administration. As mAbs are commonly manufactured into ready-to-use syringes coated with silicone oil to improve lubrication, it is important to understand how an mAb, nonionic surfactant, and silicone oil interact at the oil/water interface. In this work, we have coated a polydimethylsiloxane (PDMS) nanofilm onto an optically flat silicon substrate to facilitate the measurements of adsorption of a model mAb, COE-3, and a commercial nonionic surfactant, polysorbate 80 (PS-80), at the siliconized PDMS/water interface using spectroscopic ellipsometry and neutron reflection. Compared to the uncoated SiO2 surface (mimicking glass), COE-3 adsorption to the PDMS surface was substantially reduced, and the adsorbed layer was characterized by the dense but thin inner layer of 16 Å and an outer diffuse layer of 20 Å, indicating structural deformation. When PS-80 was exposed to the pre-adsorbed COE-3 surface, it removed 60 wt % of COE-3 and formed a co-adsorbed layer with a similar total thickness of 36 Å. When PS-80 was injected first or as a mixture with COE-3, it completely prevented COE-3 adsorption. These findings reveal the hydrophobic nature of the PDMS surface and confirm the inhibitory role of the nonionic surfactant in preventing COE-3 adsorption at the PDMS/water interface.

Preparation of highly stable and ultrasmooth chemically grafted thin films of chitosan.

Chitosan-coated surfaces are of great interest for biomedical applications (antibacterial coatings, implants, would healing, single-cell microfluidics…). However, one major limitation of chitosan-based systems is the high solubility of the polymer under acidic aqueous conditions. Herein, we describe a simple procedure to prepare extremely smooth and stable chitosan coatings. In detail, chitosan films with a low degree of N-acetylation and of thicknesses varying from 40 nm to 10 µm were grafted onto epoxy-functionalized silicon wafers via an optimized water-temperature treatment (WTT). The formation of a grafted chitosan network insoluble in acidic aqueous media (pH 3.5) was evidenced and the films were stable for at least 2 days at pH 3.5. The film morphology and the swelling behavior were characterized by atomic force microscopy (AFM) and neutron reflectivity, which showed that the film roughness was extremely low. The physical cross-linking of the films was demonstrated using infrared spectroscopy, dynamic mechanical analysis (DMA) and wide-angle X-ray scattering (WAXS). Finally, we show that the swelling behavior of such films was largely influenced by the environmental conditions, such as the pH or ionic strength of the solution.

Probing the Separation Distance between Biological Nanoparticles and Cell Membrane Mimics Using Neutron Reflectometry with Sub-Nanometer Accuracy.

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.

Lipopolysaccharides at Solid and Liquid Interfaces: Models for Biophysical Studies of the Gram-negative Bacterial Outer Membrane.

Lipopolysaccharides (LPSs) are a constitutive element of the cell envelope of Gram-negative bacteria, representing the main lipid in the external leaflet of their outer membrane (OM) lipid bilayer. These unique surface-exposed glycolipids play a central role in the interactions of Gram-negative organisms with their surrounding environment and represent a key element for protection against antimicrobials and the development of antibiotic resistance. The biophysical investigation of a wide range of different types of in vitro model membranes containing reconstituted LPS has revealed functional and structural properties of these peculiar membrane lipids, providing molecular-level details of their interaction with antimicrobial compounds. LPS assemblies reconstituted at interfaces represent a versatile tool to study the properties of the Gram-negative OM by exploiting several surface-sensitive techniques, in particular X-ray and neutron scattering, which can probe the structure of thin films with sub-nanometer resolution. This review provides an overview of different approaches employed to investigate structural and biophysical properties of LPS, focusing on studies on Langmuir monolayers of LPS at the air/liquid interface and a range of supported LPS-containing model membranes reconstituted at solid/liquid interfaces.

Lipid bilayer degradation induced by SARS-CoV-2 spike protein as revealed by neutron reflectometry.

SARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.

SARS-CoV-2 spike protein removes lipids from model membranes and interferes with the capacity of high density lipoprotein to exchange lipids.

Cholesterol has been shown to affect the extent of coronavirus binding and fusion to cellular membranes. The severity of Covid-19 infection is also known to be correlated with lipid disorders. Furthermore, the levels of both serum cholesterol and high-density lipoprotein (HDL) decrease with Covid-19 severity, with normal levels resuming once the infection has passed. Here we demonstrate that the SARS-CoV-2 spike (S) protein interferes with the function of lipoproteins, and that this is dependent on cholesterol. In particular, the ability of HDL to exchange lipids from model cellular membranes is altered when co-incubated with the spike protein. Additionally, the S protein removes lipids and cholesterol from model membranes. We propose that the S protein affects HDL function by removing lipids from it and remodelling its composition/structure.

Adhesion Process of Biomimetic Myelin Membranes Triggered by Myelin Basic Protein.

The myelin sheath-a multi-double-bilayer membrane wrapped around axons-is an essential part of the nervous system which enables rapid signal conduction. Damage of this complex membrane system results in demyelinating diseases such as multiple sclerosis (MS). The process in which myelin is generated in vivo is called myelination. In our study, we investigated the adhesion process of large unilamellar vesicles with a supported membrane bilayer that was coated with myelin basic protein (MBP) using time-resolved neutron reflectometry. Our aim was to mimic and to study the myelination process of membrane systems having either a lipid-composition resembling that of native myelin or that of the standard animal model for experimental autoimmune encephalomyelitis (EAE) which represents MS-like conditions. We were able to measure the kinetics of the partial formation of a double bilayer in those systems and to characterize the scattering length density profiles of the initial and final states of the membrane. The kinetics could be modeled using a random sequential adsorption simulation. By using a free energy minimization method, we were able to calculate the shape of the adhered vesicles and to determine the adhesion energy per MBP. For the native membrane the resulting adhesion energy per MBP is larger than that of the EAE modified membrane type. Our observations might help in understanding myelination and especially remyelination-a process in which damaged myelin is repaired-which is a promising candidate for treatment of the still mostly incurable demyelinating diseases such as MS.

Chitosan-based smart hybrid materials: a physico-chemical perspective.

Chitosan is one of the most studied cationic polysaccharides. Due to its unique characteristics of being water soluble, biocompatible, biodegradable, and non-toxic, this macromolecule is highly attractive for a broad range of applications. In addition, its complex behavior and the number of ways it interacts with different components in a system result in an astonishing variety of chitosan-based materials. Herein, we present recent advances in the field of chitosan-based materials from a physico-chemical perspective, with focus on aqueous mixtures with oppositely charged colloids, chitosan-based thin films, and nanocomposite systems. In this review, we focus our attention on the physico-chemical properties of chitosan-based materials, including solubility, mechanical resistance, barrier properties, and thermal behaviour, and provide a link to the chemical peculiarities of chitosan, such as its intrinsic low solubility, high rigidity, large charge separation, and strong tendency to form intra- and inter-molecular hydrogen bonds.

Organized Hybrid Molecular Films from Natural Phospholipids and Synthetic Block Copolymers: A Physicochemical Investigation.

In the last few years, hybrid lipid-copolymer assemblies have attracted increasing attention as possible two-dimensional (2D) membrane platforms, combining the biorelevance of the lipid building blocks with the stability and chemical tunability of copolymers. The relevance of these systems varies from fundamental studies on biological membrane-related phenomena to the construction of 2D complex devices for material science and biosensor technology. Both the fundamental understanding and the application of hybrid lipid-copolymer-supported bilayers require thorough physicochemical comprehension and structural control. Herein, we report a comprehensive physicochemical and structural characterization of hybrid monolayers at the air/water interface and of solid-supported hybrid membranes constituted by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the block copolymer poly(butadiene-b-ethyleneoxide) (PBD-b-PEO). Hybrid lipid-copolymer supported bilayers (HSLBs) with variable copolymer contents were prepared through spontaneous rupture and fusion of hybrid vesicles onto a hydrophilic substrate. The properties of the thin films and the parent vesicles were probed through dynamic light scattering (DLS), differential scanning calorimetry (DSC), optical ellipsometry, quartz crystal microbalance with dissipation monitoring (QCM-D), and confocal scanning laser microscopy (CSLM). Stable, hybrid lipid/copolymer systems were obtained for a copolymer content of 10-65 mol %. In particular, DSC and CSLM show lateral phase separation in these hybrid systems. These results improve our fundamental understanding of HSLBs, which is necessary for future applications of hybrid systems as biomimetic membranes or as drug delivery systems, with additional properties with respect to phospholipid liposomes.

Reflectometry Reveals Accumulation of Surfactant Impurities at Bare Oil/Water Interfaces.

Bare interfaces between water and hydrophobic media like air or oil are of fundamental scientific interest and of great relevance for numerous applications. A number of observations involving water/hydrophobic interfaces have, however, eluded a consensus mechanistic interpretation so far. Recent theoretical studies ascribe these phenomena to an interfacial accumulation of charged surfactant impurities in water. In the present work, we show that identifying surfactant accumulation with X-ray reflectometry (XRR) or neutron reflectometry (NR) is challenging under conventional contrast configurations because interfacial surfactant layers are then hardly visible. On the other hand, both XRR and NR become more sensitive to surfactant accumulation when a suitable scattering length contrast is generated by using fluorinated oil. With this approach, significant interfacial accumulation of surfactant impurities at the bare oil/water interface is observed in experiments involving standard cleaning procedures. These results suggest that surfactant impurities may be a limiting factor for the investigation of fundamental phenomena involving water/hydrophobic interfaces.

Structure and Conformation of Wild-Type Bacterial Lipopolysaccharide Layers at Air-Water Interfaces.

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.

Effects of oxidation on the physicochemical properties of polyunsaturated lipid membranes.

The exposure of biological membranes to reactive oxygen species (ROS) plays an important role in many pathological conditions such as inflammation, infection, or sepsis. ROS also modulate signaling processes and produce markers for damaged tissue. Lipid peroxidation, mainly affecting polyunsaturated phospholipids, results in a complex mixture of oxidized products, which may dramatically alter membrane properties. Here, we have employed a set of biophysical and surface-chemical techniques, including neutron and X-ray scattering, to study the structural, compositional, and stability changes due to oxidative stress on phospholipid bilayers composed of lipids with different degrees of polyunsaturation. In doing so, we obtained real-time information about bilayer degradation under in situ UV exposure using neutron reflectometry. We present a set of interrelated physicochemical effects, including gradual increases in area per molecule, head group and acyl chain hydration, as well as bilayer thinning, lateral phase separation, and defect formation leading to content loss upon membrane oxidation. Such effects were observed to depend on the presence of polyunsaturated phospholipids in the lipid membrane, suggesting that these may also play a role in the complex oxidation processes occurring in cells.

Structure and Composition of Native Membrane Derived Polymer-Supported Lipid Bilayers.

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.

A Versatile Method for the Distance-Dependent Structural Characterization of Interacting Soft Interfaces by Neutron Reflectometry.

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.

Sugar Surfactant Based Microemulsions at Solid Surfaces: Influence of the Oil Type and Surface Polarity.

The structure of sugar-surfactant-based bicontinuous microemulsions in the bulk and at hydrophilic and hydrophobic solid planar surfaces was studied by means of neutron scattering techniques (SANS, NR, and GISANS). In particular, the influence of the type of oil (tetradecane and methyl oleate) on the structural properties in the vicinity of surfaces was investigated at different oil-to-water ratios. In the case of hydrophilic surfaces, the analysis of the scattering length density profiles reveals an induced ordering of the oil and water domains perpendicular to the solid-liquid interface in both sets of microemulsions. At hydrophobic surfaces, differences in the near-surface ordering between microemulsions containing polar and nonpolar oils are observed.

Separation of Storage and Loss Modulus of Polyelectrolyte Multilayers on a Nanoscale: A Dynamic AFM Study.

Atomic force microscopy (AFM) is used to carry out rheology measurements on the nanoscale and to determine the mechanical properties of poly(l-lysine) (PLL)/hyaluronic acid (HA) multilayer films. Storage (G') and loss modulus (G″) of the films are calculated and compared with the values obtained from quartz crystal microbalance with dissipation monitoring measurements (QCM-D). A predominant elastic behavior independently of the applied frequencies (5-100 Hz) is observed for native HA/PLL films consisting of 36 double layer. If the layers are cross-linked, the value of G' increases by 2 orders of magnitude, while the loss modulus becomes negligible, making these films a purely elastic chemical gel. The values of G' and G'' extracted from QCM-D measurements on native films are much higher, due to the different frequency regime of the applied shear stress. However, the viscoelastic ratio from the two methods is the same and proves the elastic dominated response of the multilayer in both frequency regimes.

Construction of Compact Polyelectrolyte Multilayers Inspired by Marine Mussel: Effects of Salt Concentration and pH As Observed by QCM-D and AFM.

Biomimetic multilayers based on layer-by-layer (LbL) assembly were prepared as functional films with compact structure by incorporating the mussel-inspired catechol cross-linking. Dopamine-modified poly(acrylic acid) (PAADopa) was synthesized as a polyanion to offer electrostatic interaction with the prelayer polyethylenimine (PEI) and consecutively cross-linked by zinc to generate compact multilayers with tunable physicochemical properties. In situ layer-by-layer growth and cross-linking were monitored by a quartz crystal microbalance with dissipation (QCM-D) to reveal the kinetics of the process and the influence of Dopa chemistry. Addition of Dopa enhanced the mass adsorption and led to the formation of a more compact structure. An increase of ionic strength induced an increase in mass adsorption in the Dopa-cross-linked multilayers. This is a universal approach for coating of various surfaces such as Au, SiO2, Ti, and Al2O3. Roughness observed by AFM in both wet and dry conditions was compared to confirm the compact morphology of Dopa-cross-linked multilayers. Because of the pH sensitivity of Dopa moiety, metal-chelated Dopa groups can be turned into softer structure at higher pH as revealed by reduction of Young's modulus determined by MFP-3D AFM. A deeper insight into the growth and mechanical properties of Dopa-cross-linked polyelectrolyte multilayers was addressed in the present study. This allows a better control of these systems for bioapplications.

Transparent Aluminium Oxide Coatings of Polymer Brushes.

A novel method for the preparation of transparent Al2O3 coatings of polymers is presented. An environmental-friendly sol-gel method is employed, which implies mild conditions and low costs. A thermoresponsive brush is chosen as a model surface. X-ray photoelectron spectroscopy is used to characterize the samples during the conversion of the precursor Al(OH)3 into oxide and to prove the mildness of the protocol. The study evidences a relation between lateral homogeneity of alumina and the wettability of the polymer surface by the precursor solution, while morphology and elasticity are dominated by the polymer properties. The study of the swelling behavior of the underneath brush reveals the absence of water uptake, proving the impermeability of the alumina layer. The broad chemical and structural variety of polymers, combined with the robustness of transparent alumina films, makes these composites promising as biomedical implants, protective sheets and components for electric and optical devices.