Stephan Handschin

Mechanism of Membrane Interaction and Disruption by α-Synuclein

Parkinsons disease is a common progressive neurodegenerative condition, characterized by the deposition of amyloid fibrils as Lewy bodies in the substantia nigra of affected individuals. These insoluble aggregates predominantly consist of the protein α-synuclein. There is increasing evidence suggesting that the aggregation of α-synuclein is influenced by lipid membranes and, vice versa, the membrane integrity is severely affected by the presence of bound aggregates. Here, using the surface-sensitive imaging technique supercritical angle fluorescence microscopy and Förster resonance energy transfer, we report the direct observation of α-synuclein aggregation on supported lipid bilayers. Both the wild-type and the two mutant forms of α-synuclein studied, namely, the familiar variant A53T and the designed highly toxic variant E57K, were found to follow the same mechanism of polymerization and membrane damage. This mechanism involved the extraction of lipids from the bilayer and their clustering around growing α-synuclein aggregates. Despite all three isoforms following the same pathway, the extent of aggregation and their effect on the bilayers was seen to be variant and concentration dependent. Both A53T and E57K formed cross-β-sheet aggregates and damaged the membrane at submicromolar concentrations. The wild-type also formed aggregates in this range; however, the extent of membrane disruption was greatly reduced. The process of membrane damage could resemble part of the yet poorly understood cellular toxicity phenomenon in vivo.

Understanding nanocellulose chirality and structure-properties relationship at the single fibril level

Nanocellulose fibrils are ubiquitous in nature and nanotechnologies but their mesoscopic structural assembly is not yet fully understood. Here we study the structural features of rod-like cellulose nanoparticles on a single particle level, by applying statistical polymer physics concepts on electron and atomic force microscopy images, and we assess their physical properties via quantitative nanomechanical mapping. We show evidence of right-handed chirality, observed on both bundles and on single fibrils. Statistical analysis of contours from microscopy images shows a non-Gaussian kink angle distribution. This is inconsistent with a structure consisting of alternating amorphous and crystalline domains along the contour and supports process-induced kink formation. The intrinsic mechanical properties of nanocellulose are extracted from nanoindentation and persistence length method for transversal and longitudinal directions, respectively. The structural analysis is pushed to the level of single cellulose polymer chains, and their smallest associated unit with a proposed 2 × 2 chain-packing arrangement.

Amyloid-mediated synthesis of giant, fluorescent, gold single crystals and their hybrid sandwiched composites driven by liquid crystalline interactions

We report for the first time on the templating effect of β-lactoglobulin amyloid-like fibrils to synthesize gold single crystals of several decades of μm in dimensions. The gold single crystals were produced by reducing an aqueous solution of chloroauric acid by β-lactoglobulin amyloid protein fibrils. Atomic force microscopy, conventional and scanning transmission electron microscopy, electron diffraction and optical microscopy techniques were combined to characterize the structure of the gold crystals. The single-crystalline features of these macroscopic gold crystals are witnessed by their distinctive hexagonal and triangular shape and are confirmed by selected area electron diffraction (SAED). UV-vis absorption spectrum, recorded after a reaction time of 6h at the heating temperature of 55°C showed a surface plasmon resonance peak at 540 nm. With the increase of reaction time to 24h, the absorption spectrum peaks shift to a very broad and higher wavelength region extending up to near infrared region. Remarkably, these single crystalline gold crystals show auto fluorescence when illuminated to UV lamp. Further increase in β-lactoglobulin amyloid fibrils concentration above the isotropic-nematic transition, drives the formation of gold single crystals microplates stacking together and self-assembling into new hierarchical, layered protein-gold hybrid composites.

Complexation of beta-Lactoglobulin Fibrils and Sulfated Polysaccharides

Fibrils of β-lactoglobulin, formed by heating at pH 2, were titrated with a sulfated polysaccharide (κ-carrageenan) to determine the morphology and mechanism of complex formation at low pH. Structural information on the resultant complexes was gathered using transmission electron microscopy, atomic force microscopy, Doppler electrophoresis, and small-angle neutron scattering. Electrophoresis demonstrated that the carrageenan complexed with protein fibrils until reaching a maximum complexation efficiency at a protein/polysaccharide (r) weight ratio of 5:3. Neutron scattering and microscopy indicated an increasing formation of spherical aggregates attached along the protein fibrils with increases in the carrageenan concentration. These globular particles had an average diameter of 30 nm. Small-angle neutron scattering of these complexes could be accurately described by a form factor corresponding to multistranded twisted ribbons with spherical aggregates along their contour length, arranged in a necklace configuration.

Fibrillation of β-Lactoglobulin at Low pH in the Presence of a Complexing Anionic Polysaccharide

The influence of electrostatic complexation with κ-carrageenan was tested on the fibrillation process of β-lactoglobulin at pH 2.0. Morphology and structural development were monitored through cross correlation dynamic light scattering, transmission electron microscopy, and atomic force microscopy. Scattering indicated that noncomplexed β-lactoglobulin monomers aggregated to form fibrils after 15-90 min of heating at 90 °C. However, electrostatic protein-carrageenan complexes found in the unheated system were unchanged by the thermal process. Images and scattering results showed that carrageenan complexes slowed fibrillation kinetics, possibly through reduction in available monomer concentration. Complexes adhered to fibrils at ends and junctions in TEM images, indicating interactive affinity with the fibers, presumably as heterogeneous nucleation sites.

Twofold light and magnetic responsive behavior in nanoparticle-lyotropic liquid crystal systems.

We demonstrate the dual magnetic and light responsive nature of hybrid mesophases constituted by Fe(3)O(4) nanoparticles dispersed in lipid-based lyotropic liquid crystals (LC). When subjected to an external magnetic field in the mesophase isotropic state, the nanoparticles aggregate and orient along the magnetic field direction, and upon cooling the system through the disorder-order transition the aggregates drive the orientation of the mesophase via heterogeneous nucleation; furthermore, order-disorder transitions in the lipidic mesophase can be triggered by Fe(3)O(4)-induced photothermal effect under visible light exposure. Both the orientational order and the photothermal effect of the hybrid mesophase can be tuned by the nanoparticle content, offering a general route for controlled assembly of complex fluids with combined magnetic and light responsiveness.

Fibrillar Networks of Glycyrrhizic Acid for Hybrid Nanomaterials with Catalytic Features

Self-assembly of the naturally occurring sweetening agent, glycyrrhizic acid (GA) in water is studied by small-angle X-ray scattering and microscopic techniques. Statistical analysis on atomic force microscopy images reveals the formation of ultralong GA fibrils with uniform thickness of 2.5 nm and right-handed twist with a pitch of 9 nm, independently of GA concentration. Transparent nematic GA hydrogels are exploited to create functional hybrid materials. Two-fold and three-fold hybrids are developed by introducing graphene oxide (GO) and in situ-synthesized gold nanoparticles (Au NPs) in the hydrogel matrix for catalysis applications. In the presence of GO, the catalytic efficiency of Au NPs in the reduction of p-nitrophenol to p-aminophenol is enhanced by 2.5 times. Gold microplate single crystals are further synthesized in the GA hydrogel, expanding the scope of these hybrids and demonstrating their versatility in materials design.

Amyloid fibrils enhance transport of metal nanoparticles in living cells and induced cytotoxicity

Amyloid protein fibrils occur in vivo as pathological agents, in the case of neurodegenerative diseases, or as functional amyloids, when playing biologically vital roles. Here we show how amyloid fibrils generated from a food protein, β-lactoglobulin, can be used as nanoreactors for the synthesis of metal nanoparticles and demonstrate that the resulting hybrids can play a central role in the internalization of nanoparticles into living cells, with up to 3-fold-enhanced transport properties over pristine nanoparticles. We conjugate gold, silver, and palladium nanoparticles onto amyloid fibrils by chemical reduction, and we study their effect on dendritic and MCF7 breast cancer cells. Transmission electron microscopy indicates localization of nanoparticles inside vesicles of the cells. Flow cytometry reveals that silver nanoparticle-amyloid hybrids are cytotoxic, while gold and palladium nanoparticle-amyloid hybrids produce no notable effect on cell viability and activation status.

Gelatin-Graphene Nanocomposites with Ultralow Electrical Percolation Threshold

Gelatin-graphene conductive biopolymer nanocomposites (CPCs) with ultralow percolation threshold are designed by reducing in situ graphene oxide nanosheets with ascorbic acid and suppressing the aggregation of the graphene nanosheets. The resulting conductive nanocomposites show a record-low electrical percolation threshold of 3.3 × 10(-2) vol%, which arises from the homogeneous dispersion of the graphene nanosheets within the gelatin matrix.

Facile dispersion and control of internal structure in lyotropic liquid crystalline particles by auxiliary solvent evaporation.

Submicron sized, structured lyotropic liquid crystalline (LLC) particles, so-called hexosomes and cubosomes, are generally obtained by high energy input dispersion methods, notably ultrasonication and high-pressure emulsification. We present a method to obtain dispersions of such LLC particles with a significantly reduced energy input, by evaporation of an auxiliary volatile solvent immiscible with water, e.g. cyclohexane or limonene. The inner structure of the particles can be precisely controlled by the addition of a nonvolatile oil, such as α-tocopherol or tetradecane consistently with bulk phase diagrams,. Two different lyotropic surfactants were employed, industrial grade monolinoleine (MLO) and soy bean phosphatidylcholine (PC). The lyotropic surfactant and oil phase modifier were first dissolved in the volatile solvent to give a liquid reverse micellar (L2) phase, which requires significantly less energy input to be dispersed in an aqueous solution of secondary emulsifier compared to the corresponding gel-like bulk mesophase. The auxiliary volatile solvent was then removed from the emulsion by evaporation at room temperature, yielding LLC particles of the desired inner structure, Pn3̅m, H2, or Fd3̅m. The obtained particles were characterized by small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). Our method enables fine-tuning of the final particle size through the volatile-to-nonvolatile volume ratio and processing conditions.