Structure and Nanomechanics of Dry and Hydrated Intermediate Filament Films and Fibers Produced from Hagfish Slime Fibers.

Intermediate filaments (IFs) are known for their extensibility, flexibility, toughness, and their ability to hydrate. Using keratin-like IFs obtained from slime fibers from the invertebrate Atlantic hagfish ( Myxine glutinosa), films were produced by drop-casting and coagulation on the surface of a MgCl2 buffer. Drop-casting produced self-supporting, smooth, and dense films rich in ß-sheets (61%), whereas coagulation formed thin, porous films with a nanorough surface and a lower ß-sheet content (51%). The films hydrated and swelled immediately when immersed in water and did not dissolve. X-ray diffraction showed that the ß-crystallites remained stable upon hydration, that swelling presumably happens in the amorphous C-terminal tail-domains of the IFs, and that high salt conditions caused a denser network mesh size, suggesting polyelectrolyte behavior. Hydration resulted in a roughly 1000-fold decrease in apparent Young's modulus from 109 to 106 Pa as revealed by atomic force microscopy nanoindentation. Nanoindentation-based power-law rheology and stress-relaxation measurements indicated viscoelasticity and a soft-solid hydrogel character for hydrated films, where roughly 80% of energy is elastically stored and 20% is dissipated. By pulling coagulation films from the buffer interface, macroscopic fibers with highly aligned IF ß-crystals similar to natural hagfish fibers were produced. We propose that viscoelasticity and strong hydrogen bonding interactions with the buffer interface are crucial for the production of such long biomimetic fibers with aligned ß-sheets. This study demonstrates that hagfish fiber IFs can be reconstituted into functional biomimetic materials that are stiff when dry and retain the ability to hydrate to become soft and viscoelastic when in water.

Effect of ionic strength and seawater cations on hagfish slime formation.

The defensive slime of hagfish consists of a polyanionic mucin hydrogel that synergistically interacts with a fiber network forming a coherent and elastic hydrogel in high ionic strength seawater. In seawater, the slime deploys in less than a second entrapping large quantities of water by a well-timed thread skein unravelling and mucous gel swelling. This rapid and vast hydrogel formation is intriguing, as high ionic strength conditions generally counteract the swelling speed and ratio of polyelectrolyte hydrogels. In this work we investigate the effect of ionic strength and seawater cations on slime formation dynamics and functionality. In the absence of ionic strength skeins swell radially and unravel uncontrolled, probably causing tangling and creating a confined thread network that entraps limited water. At high ionic strength skeins unravel, but create a collapsed and dense fiber network. High ionic strength conditions therefore seem crucial for controlled skein unraveling, however not sufficient for water retention. Only the presence of naturally occurring Ca2+ or Mg2+-ions allowed for an expanded network and full water retention probably due to Ca2+-mediated vesicle rupture and cross-linking of the mucin. Our study demonstrates that hagfish slime deployment is a well-timed, ionic-strength, and divalent-cation dependent dynamic hydrogel formation process.

Hagfish slime exudate stabilization and its effect on slime formation and functionality.

Hagfish produce vast amounts of slime when under attack. The slime is the most dilute hydrogel known to date, and is a highly interesting material for biomaterial research. It forms from a glandular secrete, called exudate, which deploys upon contact with seawater. To study slime formation ex vivo and to characterize its material properties, stabilization of the sensitive slime exudate is crucial. In this study, we compared the two main stabilization methods, dispersion in high osmolarity citrate/PIPES (CP) buffer and immersion in oil, and tested the influence of time, temperature and pH on the stability of the exudate and functionality of the slime. Using water retention measurements to assess slime functionality, we found that CP buffer and oil preserved the exudate within the first 5 hours without loss of functionality. For longer storage times, slime functionality decreased for both stabilization methods, for which the breakdown mechanisms differed. Stabilization in oil likely favored temperature-sensitive osmotic-driven swelling and rupture of the mucin vesicles, causing the exudate to gel and clump. Extended storage in CP buffer resulted in an inhibited unraveling of skeins. We suggest that a water soluble protein glue, which mediates skein unraveling in functional skeins, denatures and gradually becomes insoluble during storage in CP buffer. The breakdown was accentuated when the pH of the CP buffer was raised from pH 6.7 to pH 8.5, probably caused by increased denaturation of the protein glue or by inferior vesicle stabilization. However, when fresh exudate was mixed into seawater or phosphate buffer at pH 6-9, slime functionality was not affected, showing pH insensitivity of the slime formation around a neutral pH. These insights on hagfish exudate stabilization mechanisms will support hagfish slime research at a fundamental level, and contribute to resolve the complex mechanisms of skein unraveling and slime formation.

Mechanically Enhanced Liquid Interfaces at Human Body Temperature Using Thermosensitive Methylated Nanocrystalline Cellulose.

The mechanical performance of materials at oil/water interfaces after consumption is a key factor affecting hydrophobic drug release. In this study, we methylated the surface of nanocrystalline cellulose (NCC) by mercerization and dimethyl sulfate exposure to produce thermosensitive biopolymers. These methylated NCC (metNCC) were used to investigate interfacial thermogelation at air/water and medium-chain triglyceride (MCT)/water interfaces at body temperature. In contrast to bulk fluid dynamics, elastic layers were formed at room temperature, and elasticity increased significantly at body temperature, which was measured by interfacial shear and dilatational rheology in situ. This unique phenomenon depends on solvent quality, temperature, and polymer concentration at interfaces. Thus, by adjusting the degree of hydrophobicity of metNCC, the interfacial elasticity and thermogelation of the interfaces could be varied. In general, these new materials (metNCC) formed more brittle interfacial layers compared to commercial methylcellulose (MC A15). Thermogelation of methylcellulose promotes attractive intermolecular forces, which were reflected in a change in self-assembly of metNCC at the interface. As a consequence, layer thickness and density increased as a function of temperature. These effects were measured by atomic force microscopy (AFM) images of the displaced interface and confirmed by neutron reflection. The substantial structural and mechanical change of methylcellulose interfaces at body temperature represents a controllable encapsulation parameter allowing optimization of lipid-based drug formulations.

Tailored interfacial rheology for gastric stable adsorption layers.

Human lipid digestion begins at the interface of oil and water by interfacial adsorption of lipases. Tailoring the available surface area for lipase activity can lead to specific lipid sensing in the body, thus, tailored satiety hormone release. In this study we present biopolymer layers at the MCT-oil/water interface with different stabilities under human gastric environment (37 °C, pH 2, pepsin). Physicochemical changes and enzymatic degradation of interfacial layers were monitored online by interfacial shear rheology. We show the weakening of ß-lactoglobulin (ß-lg) layers at body temperature and acidification and their hydrolysis by pepsin. If sufficient concentrations of nanocrystalline cellulose (NCC) are given to an existing ß-lg layer, this weakening is buffered and the proteolysis delayed. A synergistic, composite layer is formed by adding methylated NCC to the ß-lg layer. This layer thermogels at body temperature and resists hydrolysis by pepsin. Coexistence of these two emulsifiers at the air/water interface is evidenced by neutron reflectometry measurements, where morphological information are extracted. The utilized layers and their analysis provide knowledge of physicochemical changes during in vitro digestion of interfaces, which promote functional food formulations.

Mechanism of bubble coalescence induced by surfactant covered antifoam particles.

Mechanism of inter-bubble coalescence by an aqueous fatty alcohol particle suspension antifoam containing a nonionic surfactant has been investigated. By observing visually two colliding air bubbles in a liquid pool in the presence of the antifoam, a four-step mechanism is identified. The role of the surfactant in the antifoam is, for the first time, proposed. A surface tension gradient due to the local surfactant concentration difference enables a surfactant laden hydrophobic particle located on bubble surface to move from the periphery of a liquid film between two colliding air bubbles to their region of contact. Drop volume tensiometry and macroscopic foam column experiments are used to further prove this observation. Subsequently, the particle bridges and dewets the bubbles resulting in film rupture. The rate of drainage of the liquid film depends on the particle hydrophobicity, which necessitates complete surfactant desorption from particle surface. This is corroborated experimentally by Wilhelmy plate tensiometry. In addition, cryo-scanning electron and atomic force microscopy are used to determine the particle shape and the force for its entry into the bubble.

Influence of pH on colloidal properties and surface activity of polyglycerol fatty acid ester vesicles.

Certain polyglycerol esters of fatty acids (PGE) form dispersions of uni- or multilamellar vesicles in dilute aqueous solution. These self-assembled aggregates reduce the surface-activity of PGE monomers such that interfacial films may take several hours to form. This is undesirable for processes, which rely on rapid surfactant adsorption, for example foaming. In the present work, we study the effect of pH on the colloidal (size distribution, morphology, surface charge) and interfacial (adsorption kinetics) properties of a commercial, non-purified PGE. Using dynamic light scattering, zeta-potential measurements and cryo-SEM, we show that changing the pH of the dispersion media can cause agglomeration and eventually osmotic rupture of PGE vesicles. The change in dispersion state also impacts the adsorption behavior at the water surface. Direct evidence that destabilized vesicle dispersion are more surface-active is provided by comparing the dynamic surface tension of solutions of different pH. The faster adsorption kinetics at low pH correlate with a remarkably increased foaming power. We suggest that an osmotic shock induced by changes in pH causes vesicles to deform and partially open, so that their hydrocarbon core is exposed to the dispersion media. This energetically unfavorable condition promotes the hydrophobically driven adsorption of surfactant monomers at surfaces and hence stimulates the foaming ability.

Nonionic block copolymer antifoams.

Aqueous dispersions of alkoxylated alcohol block copolymer (BCP) drops are investigated as antifoams. A model aqueous nonionic surfactant solution of Polysorbate 20 and an industrial white water suspension are used as foaming systems. Visual evidence obtained using a two-bubble technique involving a CCD camera coupled with high magnification lenses clearly revealed the role of BCP droplets in the bubble coalescence process. The enhancement of bubble coalescence decreased as the temperature increased from 25 to 60 degrees C, which is due to the corresponding decrease in the rigidity associated with the weak interfacial structure and reduced viscosity of the BCP drops. The antifoaming efficiency measured in the macroscopic recirculation foam column increased with temperature from about 13 to 26 degrees C (attaining a maximum) and decreased as temperature increased further. Oscillatory thermo-rheometric measurements showed a sudden increase in the storage modulus (G') by several orders of magnitude, indicating gel formation initiated at about 13 degrees C and having a maximum at around 26 degrees C for an aqueous solution of the BCP above a critical concentration of around 20 wt %. Results obtained using small-angle X-ray scattering, micro-differential scanning calorimetry, and proton nuclear magnetic resonance confirmed the existence of ordered gel-like structures. Furthermore, macroscopic tests using a sparged air foam column showed a significant increase in antifoaming efficiency when highly hydrophobic particles are embedded in the BCP drops dispersed in water.

Local film thinning due to concentration gradient of an insoluble surfactant at an interface.

The local thinning of a viscous liquid film on a substrate driven by a surface (or interfacial) tension gradient due to a concentration gradient of a monolayer of an insoluble surfactant initially non-uniformly distributed at a liquid interface relevant to chemical engineering, biomedical and other applications is investigated. A simple model is presented for the temporal evolution of the profiles of radial variation in the thickness of a thin liquid film, the effects of gravity and capillarity due to deformation of the interface in slowing down the film thinning process being allowed. As time increases, the surfactant spreads and the radius of its front increases inversely with decrease in the two-third power of the film thickness at the center. The model describes well not only the published experimental results but also those obtained by other authors using numerical simulations of a set of coupled partial differential equations.