Assembling responsive microgels at responsive lipid membranes.

Directed colloidal self-assembly at fluid interfaces can have a large impact in the fields of nanotechnology, materials, and biomedical sciences. The ability to control interfacial self-assembly relies on the fine interplay between bulk and surface interactions. Here, we investigate the interfacial assembly of thermoresponsive microgels and lipogels at the surface of giant unilamellar vesicles (GUVs) consisting of phospholipids bilayers with different compositions. By altering the properties of the lipid membrane and the microgel particles, it is possible to control the adsorption/desorption processes as well as the organization and dynamics of the colloids at the vesicle surface. No translocation of the microgels and lipogels through the membrane was observed for any of the membrane compositions and temperatures investigated. The lipid membranes with fluid chains provide highly dynamic interfaces that can host and mediate long-range ordering into 2D hexagonal crystals. This is in clear contrast to the conditions when the membranes are composed of lipids with solid chains, where there is no crystalline arrangement, and most of the particles desorb from the membrane. Likewise, we show that in segregated membranes, the soft microgel colloids form closely packed 2D crystals on the fluid bilayer domains, while hardly any particles adhere to the more solid bilayer domains. These findings thus present an approach for selective and controlled colloidal assembly at lipid membranes, opening routes toward the development of tunable soft materials.

Interfacial properties of lipid sponge-like nanoparticles and the role of stabilizer on particle structure and surface interactions.

The advantage of using nonlamellar lipid liquid crystalline phases has been demonstrated in many applications, such as drug delivery, protein encapsulation and crystallisation. We have recently reported that a mixture of mono- and diglycerides is able to form sponge-like nanoparticles (L3-NPs) with large enough aqueous pores to encapsulate macromolecules such as proteins. Here we use small angle neutron scattering (SANS) to reveal morphology, structural and chemical composition of these polysorbate 80 (P80) stabilized sponge phase nanoparticles, not previously known. Our results suggest that L3-NPs have a core-shell sphere structure, with a shell rich in P80. It was also found that even if P80 is mostly located on the surface, it also contributes to the formation of the inner sponge phase structure. An important aspect for the application and colloidal stability of these particles is their interfacial properties. Therefore, the interfacial behaviour of the nanoparticles on hydrophilic silica was revealed by Quartz crystal microbalance with dissipation (QCM-D) and neutron reflectivity (NR). Adsorption experiments reveal the formation of a thin lipid layer, with the dimension corresponding to a lipid bilayer after L3-NPs are in contact with hydrophilic silica. This suggests that the diglycerol monoleate/Capmul GMO-50/P80 particles reorganize themselves on this surface, probably due to interactions between P80 head group and SiO2.

Nanowires for Biosensing: Lightguiding of Fluorescence as a Function of Diameter and Wavelength.

Semiconductor nanowires can act as nanoscaled optical fibers, enabling them to guide and concentrate light emitted by surface-bound fluorophores, potentially enhancing the sensitivity of optical biosensing. While parameters such as the nanowire geometry and the fluorophore wavelength can be expected to strongly influence this lightguiding effect, no detailed description of their effect on in-coupling of fluorescent emission is available to date. Here, we use confocal imaging to quantify the lightguiding effect in GaP nanowires as a function of nanowire geometry and light wavelength. Using a combination of finite-difference time-domain simulations and analytical approaches, we identify the role of multiple waveguide modes for the observed lightguiding. The normalized frequency parameter, based on the step-index approximation, predicts the lightguiding ability of the nanowires as a function of diameter and fluorophore wavelength, providing a useful guide for the design of optical biosensors based on nanowires.

Supported Fluid Lipid Bilayer as a Scaffold to Direct Assembly of RNA Nanostructures.

RNA architectonics offers the possibility to design and assemble RNA into specific shapes, such as nanoscale 3D solids or nanogrids. Combining the minute size of these programmable shapes with precise positioning on a surface further enhances their potential as building blocks in nanotechnology and nanomedicine. Here we describe a bottom-up approach to direct the arrangement of nucleic acid nanostructures by using a supported fluid lipid bilayer as a surface scaffold. The strong attractive electrostatic interactions between RNA polyanions and cationic lipids promote RNA adsorption and self-assembly. Protocol steps for the characterization of assembled RNA complexes via several complementary methods (QCM-D, ellipsometry, confocal fluorescence microscopy, AFM) are also provided. Due to their tunable nature, lipid bilayers can be used to organize RNA laterally on the micrometer scale and thus facilitate the building of more complex 3D structures. The bilayer-based approach can be extended to other programmable RNA or DNA objects to construct intricate structures, such as 2D grids or 3D cages, with high precision.

Non-lamellar lipid assembly at interfaces: controlling layer structure by responsive nanogel particles.

Biological membranes do not only occur as planar bilayer structures, but depending on the lipid composition, can also curve into intriguing three-dimensional structures. In order to fully understand the biological implications as well as to reveal the full potential for applications, e.g. for drug delivery and other biomedical devices, of such structures, well-defined model systems are required. Here, we discuss the formation of lipid non-lamellar liquid crystalline (LC) surface layers spin-coated from the constituting lipids followed by hydration of the lipid layer. We demonstrate that hybrid lipid polymer films can be formed with different properties compared with the neat lipid LC layers. The nanostructure and morphologies of the lipid films formed reflect those in the bulk. Most notably, mixed lipid layers, which are composed of glycerol monooleate and diglycerol monooleate with poly(N-isopropylacrylamide) nanogels, can form films of reverse cubic phases that are capable of responding to temperature stimulus. Owing to the presence of the nanogel particles, changing the temperature not only regulates the hydration of the cubic phase lipid films, but also the lateral organization of the lipid domains within the lipid self-assembled film. This opens up the possibility for new nanostructured materials based on lipid-polymer responsive layers.

Interfacial properties of POPC/GDO liquid crystalline nanoparticles deposited on anionic and cationic silica surfaces.

Reversed lipid liquid crystalline nanoparticles (LCNPs) of the cubic micellar (I2) phase have high potential in drug delivery applications due to their ability to encapsulate both hydrophobic and hydrophilic drug molecules. Their interactions with various interfaces, and the consequences for the particle structure and integrity, are essential considerations in their effectiveness as drug delivery vehicles. Here, we have studied LCNPs formed of equal fractions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and glycerol dioleate in the presence of different fractions of the stabilizer Polysorbate 80. We have used a combination of ellipsometry, quartz crystal microbalance with dissipation monitoring and neutron reflectometry to reveal the structure and composition of the adsorbed layer on both anionic silica and cationic (aminopropyltriethoxysilane) silanized surfaces. For both types of surfaces, there is a spread near-surface layer comprising lipid and polymer as well as a sparse coverage of intact particles. The composition of the near-surface layer is very close to that of the particles, in contrast to the lipid bilayer observed with related systems. The interaction is stronger for cationic than anionic surfaces, which is rationalized in terms of the negative zeta potential of the LCNPs. The work shows that the attachment of and spreading from LCNPs is influenced by the properties of the surface, the internal structure, composition and stability of the particles as well as the nature of the stabilizer.

Diffusion through Pig Gastric Mucin: Effect of Relative Humidity.

Mucus covers the epithelium found in all intestinal tracts, where it serves as an important protecting barrier, and pharmaceutical drugs administrated by the oral, rectal, vaginal, ocular, or nasal route need to penetrate the mucus in order to reach their targets. Furthermore, the diffusion in mucus as well as the viscosity of mucus in the eyes, nose and throat can change depending on the relative humidity of the surrounding air. In this study we have investigated how diffusion through gels of mucin, the main protein in mucus, is affected by changes in ambient relative humidity (i.e. water activity). Already a small decrease in water activity was found to give rise to a significant decrease in penetration rate through the mucin gel of the antibacterial drug metronidazole. We also show that a decrease in water activity leads to decreased diffusion rate in the mucin gel for the fluorophore fluorescein. This study shows that it is possible to alter transport rates of molecules through mucus by changing the water activity in the gel. It furthermore illustrates the importance of considering effects of the water activity in the mucosa during development of potential pharmaceuticals.

Surface nanostructures for fluorescence probing of supported lipid bilayers on reflective substrates.

The fluorescence interference contrast (FLIC) effect prevents the use of fluorescence techniques to probe the continuity and fluidity of supported lipid bilayers on reflective materials due to a lack of detectable fluorescence. Here we show that adding nanostructures onto reflective surfaces to locally confer a certain distance between the deposited fluorophores and the reflecting surface enables fluorescence detection on the nanostuctures. The nanostructures consist of either deposited nanoparticles or epitaxial nanowires directly grown on the substrate and are designed such that they can support a lipid bilayer. This simple method increases the fluorescence signal sufficiently to enable bilayer fluorescence detection and to observe the recovery of fluorescence after photobleaching in order to assess lipid bilayer formation on any reflective surface.

Model-free estimation of the effective correlation time for C-H bond reorientation in amphiphilic bilayers: (1)H-(13)C solid-state NMR and MD simulations.

Molecular dynamics (MD) simulations give atomically detailed information on structure and dynamics in amphiphilic bilayer systems on timescales up to about 1 µs. The reorientational dynamics of the C-H bonds is conventionally verified by measurements of (13)C or (2)H nuclear magnetic resonance (NMR) longitudinal relaxation rates R1, which are more sensitive to motional processes with correlation times close to the inverse Larmor frequency, typically around 1-10 ns on standard NMR instrumentation, and are thus less sensitive to the 10-1000 ns timescale motion that can be observed in the MD simulations. We propose an experimental procedure for atomically resolved model-free estimation of the C-H bond effective reorientational correlation time τe, which includes contributions from the entire range of all-atom MD timescales and that can be calculated directly from the MD trajectories. The approach is based on measurements of (13)C R1 and R1ρ relaxation rates, as well as (1)H-(13)C dipolar couplings, and is applicable to anisotropic liquid crystalline lipid or surfactant systems using a conventional solid-state NMR spectrometer and samples with natural isotopic composition. The procedure is demonstrated on a fully hydrated lamellar phase of 1-palmitoyl-2-oleoyl-phosphatidylcholine, yielding values of τe from 0.1 ns for the methyl groups in the choline moiety and at the end of the acyl chains to 3 ns for the g1 methylene group of the glycerol backbone. MD simulations performed with a widely used united-atom force-field reproduce the τe-profile of the major part of the acyl chains but underestimate the dynamics of the glycerol backbone and adjacent molecular segments. The measurement of experimental τe-profiles can be used to study subtle effects on C-H bond reorientational motions in anisotropic liquid crystals, as well as to validate the C-H bond reorientation dynamics predicted in MD simulations of amphiphilic bilayers such as lipid membranes.

Association of anionic surfactant and physisorbed branched brush layers probed by neutron and optical reflectometry.

Pre-adsorbed branched brush layers were formed on silica surfaces by adsorption of a diblock copolymer consisting of a linear cationic block and an uncharged bottle-brush block. The charge of the silica surface was found to affect the adsorption, with lower amounts of the cationic polyelectrolyte depositing on less charged silica. Cleaning under basic conditions rendered surfaces more negatively charged (more negative zeta-potential) than acid cleaning and was therefore used to increase polyelectrolyte adsorption. The structure of adsorbed layers of the diblock copolymer was as determined by neutron reflectometry found to be about 70 nm thick and very water rich (97%). Interactions between the anionic surfactant sodium dodecylsulfate (SDS) and such pre-adsorbed diblock polymer layers were studied by neutron reflectometry and by optical reflectometry. Optical reflectometry was also used for deducing interactions between the individual blocks of the diblock copolymer and SDS at the silica/aqueous interface. We find that SDS is readily incorporated in the diblock copolymer layer at low SDS concentrations, and preferentially co-localized with the cationic block of the polymer next to the silica surface. At higher SDS concentrations some desorption of polyelectrolyte/surfactant complexes takes place.

Non-lamellar lipid liquid crystalline structures at interfaces.

The self-assembly of lipids leads to the formation of a rich variety of nano-structures, not only restricted to lipid bilayers, but also encompassing non-lamellar liquid crystalline structures, such as cubic, hexagonal, and sponge phases. These non-lamellar phases have been increasingly recognized as important for living systems, both in terms of providing compartmentalization and as regulators of biological activity. Consequently, they are of great interest for their potential as delivery systems in pharmaceutical, food and cosmetic applications. The compartmentalizing nature of these phases features mono- or bicontinuous networks of both hydrophilic and hydrophobic domains. To utilize these non-lamellar liquid crystalline structures in biomedical devices for analyses and drug delivery, it is crucial to understand how they interact with and respond to different types of interfaces. Such non-lamellar interfacial layers can be used to entrap functional biomolecules that respond to lipid curvature as well as the confinement. It is also important to understand the structural changes of deposited lipid in relation to the corresponding bulk dispersions. They can be controlled by changing the lipid composition or by introducing components that can alter the curvature or by deposition on nano-structured surface, e.g. vertical nano-wire arrays. Progress in the area of liquid crystalline lipid based nanoparticles opens up new possibilities for the preparation of well-defined surface films with well-defined nano-structures. This review will focus on recent progress in the formation of non-lamellar dispersions and their interfacial properties at the solid/liquid and biologically relevant interfaces.

Assembly of RNA nanostructures on supported lipid bilayers.

The assembly of nucleic acid nanostructures with controlled size and shape has large impact in the fields of nanotechnology, nanomedicine and synthetic biology. The directed arrangement of nano-structures at interfaces is important for many applications. In spite of this, the use of laterally mobile lipid bilayers to control RNA three-dimensional nanostructure formation on surfaces remains largely unexplored. Here, we direct the self-assembly of RNA building blocks into three-dimensional structures of RNA on fluid lipid bilayers composed of cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or mixtures of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and cationic sphingosine. We demonstrate the stepwise supramolecular assembly of discrete building blocks through specific and selective RNA-RNA interactions, based on results from quartz crystal microbalance with dissipation (QCM-D), ellipsometry, fluorescence recovery after photobleaching (FRAP) and total internal reflection fluorescence microscopy (TIRF) experiments. The assembly can be controlled to give a densely packed single layer of RNA polyhedrons at the fluid lipid bilayer surface. We show that assembly of the 3D structure can be modulated by sequence specific interactions, surface charge and changes in the salt composition and concentration. In addition, the tertiary structure of the RNA polyhedron can be controllably switched from an extended structure to one that is dense and compact. The versatile approach to building up three-dimensional structures of RNA does not require modification of the surface or the RNA molecules, and can be used as a bottom-up means of nanofabrication of functionalized bio-mimicking surfaces.

Enhanced laminin adsorption on nanowires compared to flat surfaces.

Semiconductor nanowires are widely used to interface living cells, and numerous nanowire-based devices have been developed to manipulate or sense cell behavior. We have, however, little knowledge on the nature of the cell-nanowire interface. Laminin is an extracellular matrix protein promoting cell attachment and growth. Here, we used a method based on fluorescence microscopy and measured the relative amount of laminin adsorbed on nanowires compared to flat surfaces. The amount of adsorbed laminin per surface area is up to 4 times higher on 55nm diameter gallium phosphide nanowires compared to the flat gallium phosphide surface between the nanowires. We show that this enhanced adsorption on nanowires cannot be attributed to electrostatic effects, nor to differences in surface chemistry, but possibly to pure geometrical effects, as increasing the nanowire diameter results in a decreased amount of adsorbed protein. The increased adsorption of laminin on nanowires may explain the exceptionally beneficial properties of nanowire substrates for cellular growth reported in the literature since laminin is often used as surface coating prior to cell cultures in order to promote cell growth, and also because primary cell suspensions contain endogenous laminin.

Modulation of dipalmitoylphosphatidylcholine monolayers by dimethyl sulfoxide.

The action of the penetration-enhancing agent, dimethyl sulfoxide (DMSO), on phospholipid monolayers was investigated at the air-water interface using a combination of experimental techniques and molecular dynamics simulations. Brewster angle microscopy revealed that DPPC monolayers remained laterally homogeneous at subphase concentrations up to a mole fraction of 0.1 DMSO. Neutron reflectometry of the monolayers in combination with isotopic substitution enabled the determination of solvent profiles as a function of distance perpendicular to the interface for the different DMSO subphase concentrations. These experimental results were compared to those obtained from molecular dynamic (MD) simulations of the corresponding monolayer systems. There was excellent agreement found between the MD-derived reflectivity curves and the measured data for all of the H/D contrast variations investigated. The MD provide a detailed description of the distribution of water and DMSO molecules around the phosphatidylcholine headgroup, and how this distribution changes with increasing DMSO concentrations. Significantly, the measurements and simulations that are reported here support the hypothesis that DMSO acts by dehydrating the phosphatidylcholine headgroup, and as such provide the first direct evidence that it does so primarily by displacing water molecules bound to the choline group.

Fluid and highly curved model membranes on vertical nanowire arrays.

Sensing and manipulating living cells using vertical nanowire devices requires a complete understanding of cell behavior on these substrates. Changes in cell function and phenotype are often triggered by events taking place at the plasma membrane, the properties of which are influenced by local curvature. The nanowire topography can therefore be expected to greatly affect the cell membrane, emphasizing the importance of studying membranes on vertical nanowire arrays. Here, we used supported phospholipid bilayers as a model for biomembranes. We demonstrate the formation of fluid supported bilayers on vertical nanowire forests using self-assembly from vesicles in solution. The bilayers were found to follow the contours of the nanowires to form continuous and locally highly curved model membranes. Distinct from standard flat supported lipid bilayers, the high aspect ratio of the nanowires results in a large bilayer surface available for the immobilization and study of biomolecules. We used these bilayers to bind a membrane-anchored protein as well as tethered vesicles on the nanowire substrate. The nanowire-bilayer platform shown here can be expanded from fundamental studies of lipid membranes on controlled curvature substrates to the development of innovative membrane-based nanosensors.

Calcium-mediated binding of DNA to 1,2-distearoyl-sn-glycero-3-phosphocholine-containing mixed lipid monolayers.

The calcium-mediated interaction of DNA with monolayers of the non-toxic, zwitterionic phospholipid, 1,2-distearoyl-sn-glycero-3-phosphocholine when mixed with 50 mol% of a second lipid, either the zwitteronic 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or neutral cholesterol was investigated using a combination of surface pressure-area isotherms, Brewster angle microscopy, external reflectance Fourier transform infrared spectroscopy and specular neutron reflectivity in combination with contrast variation. When calcium and DNA were both present in the aqueous subphase, changes were observed in the compression isotherms as well as the surface morphologies of the mixed lipid monolayers. In the presence of calcium and DNA, specular neutron reflectivity showed that directly underneath the head groups of the lipids comprising the monolayers, DNA occupied a layer comprising approximately 13 and 18% v/v DNA for the 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and cholesterol-containing monolayers, respectively. The volume of the corresponding layer for 1,2-distearoyl-sn-glycero-3-phosphocholine only containing monolayers was ∼15% v/v DNA. Furthermore regardless of the presence and nature of the second lipid and the surface pressure of the monolayer, the specular neutron reflectivity experiments showed that the DNA-containing layer was 20-27 Šthick, suggesting the presence of a well-hydrated layer of double-stranded DNA. External reflectance Fourier transform infrared studies confirmed the presence of double stranded DNA, and indicated that the strands are in the B-form conformation. The results shed light on the interaction between lipids and nucleic acid cargo as well as the role of a second lipid in lipid-based carriers for drug delivery.

Tunable adsorption of soft colloids on model biomembranes.

A simple procedure is developed to probe in situ the association between lipid bilayers and colloidal particles. Here, a one-step method is applied to generate giant unilamellar 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles (GUVs) by application of an alternating electric field directly in the presence of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) microgels. We demonstrate that the soft PNIPAM microgel particles act as switchable stabilizers for lipid membranes. The change of the particle conformation from the swollen to the collapsed state enables the reversible control of the microgel adsorption as a function of temperature. At 20 °C, the swollen and hydrophilic soft microgel particles adsorb evenly and densely pack in 2D hexagonal arrays at the DOPC GUV surfaces. In contrast, at 40 °C, that is, above the volume phase transition temperature (TVPT = 32 °C) of the PNIPAM microgels, the collapsed and more hydrophobic particles partially desorb and self-organize into domains at the GUV/GUV interfaces. This study shows that thermoresponsive PNIPAM microgels can be used to increase and control the stability of lipid vesicles where the softness and deformability of these types of particles play a major role. The observed self-assembly, where the organization and position of the particles on the GUV surface can be controlled "on demand", opens new routes for the design of nanostructured materials.

On the solvation structure of dimethylsulfoxide/water around the phosphatidylcholine head group in solution.

The solution structure of the phosphocholine (PC) head group in 1,2-dipropionyl-sn-glycero-3-phosphocholine (C(3)-PC) in 30 mol. % dimethylsulfoxide (DMSO)-water solutions has been determined by using neutron diffraction enhanced with isotopic substitution in combination with computer simulation techniques. By investigating the atomic scale hydration structure around the PC head group, a unique description of the displacement of water molecules by DMSO molecules is detailed around various locations of the head group. Specifically, DMSO molecules were found to be the most prevalent around the onium portion of the head group, with the dipoles of the DMSO molecules being aligned where the negatively charged oxygen can interact strongly with the positively charged lipid group. The phosphate group is also partially dehydrated by the presence of the DMSO molecules. However, around this group the bulkier positive end of the DMSO dipole is interacting with negatively charged groups of the lipid head group, the DMSO layer shows no obvious ordering as it cannot form hydrogen bonds with the oxygen atoms in the PO(4) group such as water molecules can. Interestingly, DMSO-water contacts have also increased in the presence of the lipid molecule relative to DMSO-water contacts observed in pure DMSO/water solutions at similar concentrations.