Michael F. Butler

Rational design and application of responsive |[alpha]|-helical peptide hydrogels

Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medicine, such as the controlled delivery and release of cells, cosmetics and drugs; and as supports for cell growth and tissue engineering1. Rational peptide design and engineering are emerging as promising new routes to such functional biomaterials2-4. Here we present the first examples of rationally designed and fully characterized self-assembling hydrogels based on standard linear peptides with purely α-helical structures, which we call hydrogelating self-assembling fibres (hSAFs). These form spanning networks of α-helical fibrils that interact to give self-supporting physical hydrogels of >99% water content. The peptide sequences can be engineered to alter the underlying mechanism of gelation and, consequently, the hydrogel properties. Interestingly, for example, those with hydrogen-bonded networks melt upon heating, whereas those formed via hydrophobic interactions strengthen when warmed. The hSAFs are dual-peptide systems that only gel on mixing, which gives tight control over assembly5. These properties raise possibilities for using the hSAFs as substrates in cell culture. We have tested this in comparison with the widely used Matrigel substrate, and demonstrate that, like Matrigel, hSAFs support both growth and differentiation of rat adrenal pheochromocytoma cells for sustained periods in culture.

A new method for maintaining homogeneity during liquid–hydrogel transitions using low molecular weight hydrogelators

We demonstrate a generic new approach to produce homogeneous and reproducible hydrogels from low molecular weight hydrogelators using the controlled hydrolysis of glucono-δ-lactone (GdL). GdL slowly hydrolyses in water to give gluconic acid, which controllably lowers the pH. This hydrolysis is slower than the rate of dissolution; hence uniform pH change throughout the sample is possible. This results in homogeneous hydrogels that are unaffected by their shear or mixing history. A further advantage of this method is that it allows the gelation process to be monitored, giving further insight into the mechanism by which gelation occurs.

Time resolved simultaneous small- and wide-angle X-ray scattering during polyethylene deformation. II. Cold drawing of linear polyethylene

The cold drawing behaviour of a range of unoriented linear polyethylenes was investigated using the technique of simultaneous small- and wide-angle X-ray scattering during deformation. The influences of molecular weight and thermal history were studied. The role of both variables was found to be highly important since by altering the percentage crystallinity they influenced the relative amounts of inter- and intra-lamellar deformation. The micromechanical deformation processes governing the different regions of the load-extension curve were found to be the same as for cold-drawn ethylene-α-olefin copolymers, demonstrating the fundamental similarity between all types of unoriented polyethylene PE.

Time resolved simultaneous small- and wide-angle X-ray scattering during polyethylene deformation : 1. Cold drawing of ethylene-α-olefin copolymers

The cold drawing behaviour of a range of polyethylene copolymers was investigated using the technique of simultaneous small- and wide-angle X-ray scattering (SAXS and WAXS, respectively) during deformation. The influence of branch amount, branch length and lamellar population were studied. Branch length was found to be unimportant. Increasing branch content lowered the percentage crystallinity which resulted in an increase in the importance of interlamellar deformation mechanisms. Double yield points were observed in some samples. Molecular and lamellar deformation was totally reversible up to the first yield point, and was apportioned to the activation of fine chain slip. After the second yield point, for which coarse chain slip leading to lamellar fragmentation was held responsible, deformation was irreversible.

Templating silica nanostructures on rationally designed self-assembled peptide fibers.

Nature presents exquisite examples of templating hard, functional inorganic materials on soft, self-assembled organic substrates. An ability to mimic and control similar processes in the laboratory would increase our understanding of fundamental science, and may lead to potential applications in the broad arena of bionanotechnology. Here we describe how self-assembled, alpha-helix-based peptide fibers of de novo design can promote and direct the deposition of silica from silicic acid solutions. The peptide substrate can be removed readily through proteolysis, or other facile means to render silica nanotubes. Furthermore, the resulting silica structures, which span the nanometer to micrometer range, can themselves be used to template the deposition of the cationic polyelectrolyte, poly-(diallyldimethylammonium chloride). Finally, the peptide-based substrates can be engineered prior to silicification to alter the morphology and mechanical properties of the resulting hybrid and tubular materials.

The ductile–brittle transition of irradiated isotactic polypropylene studied using simultaneous small angle X-ray scattering and tensile deformation

Abstract This research concerns the micromechanical deformation mechanisms of irradiated and non-irradiated isotactic polypropylene (iPP), studied as a function of temperature above the glass transition. Several deformation mechanisms were identified and included lamellar separation, shear, stable and unstable fibrillated deformation and cavitation. The ductile–brittle transition rises dramatically with irradiation, while the glass transition shows only a small increase. This observation is explained by irradiation, through chain scission and cross-linking, having a dominant effect on large-scale plastic deformation, and a lesser effect on the deformation which relies on the amorphous phase alone.

Impact of mechanism of formation on encapsulation in block copolymer vesicles

Vesicles prepared from block copolymers have been mooted for the encapsulation of water-soluble molecules. This is because the membranes of polymer vesicles have been shown to be more stable than those in vesicles formed from lipids, with the membrane properties being tuned by the length and nature of the hydrophobic block in the polymer. The generally accepted mechanisms of vesicle formation involve either wrap-up of a lamellar sheet or formation via a sequence of micelle to worm to disks to vesicles. These should lead to efficient encapsulation. Alternatively, a method involving phase separation followed by re-structuring has been recently suggested. Here, we show that this final mechanism holds for vesicles formed from a PEO-b-PDEAMA copolymer by a pH switch and that this mechanism leads to highly inefficient encapsulation on vesicle formation.

Phase separation in gelatin/dextran and gelatin/maltodextrin mixtures

Abstract Phase separation mechanisms and kinetics in quiescent and shear conditions were studied using small-angle light scattering, optical polarimetry and confocal laser scanning microscopy in the gelatin/maltodextrin and gelatin/dextran systems. In the former system the temperature quench caused phase separation, which was studied in the gelled and liquid states, whereas in the latter system phase separation was triggered by the conformational ordering of the gelatin molecules and could only be studied when the system gelled. In both systems the different phase separation mechanisms of nucleation and growth and spinodal decomposition were identified from the different behaviour of structure function measured by light scattering. In the liquid state coarsening of the microstructure occurred by droplet coalescence that was accelerated by hydrodynamic effects when the droplets reached a certain size. Gelation hindered, but did not prevent coarsening. Reduced coarsening rates were measured in the gelled systems. In most cases the phase separation kinetics were faster than the gelation kinetics, and the system rapidly evolved into the late stages of phase separation that were characterised by a well-defined morphology with sharp interfaces. For sufficiently rapid ordering kinetics, corresponding to deep quenches, in the gelatin/dextran systems, however, it was possible to trap the microstructure in the early stages of phase separation while the interfaces were still diffuse. When the phase-separated liquid gelatin/maltodextrin system was sheared, coarsening was accelerated at low shear rates due to increased rates of droplet coalescence. At higher shear rates, stable elongated structures were formed. At one particular shear rate (approximately 1 s−1), the rates of break-up and coalescence were balanced and a monodisperse size distribution of elongated droplets was formed.

A study of the molecular relaxations in solid starch using dielectric spectroscopy

Abstract A number of relaxations have been observed via dielectric spectroscopy, and possible molecular origins assigned, in a range of gelatinised and granular solid starches. At low temperatures (in the region of −120°C) two relaxations occur. The evidence indicated that these were due to small motions of the chain backbone and rotation of methylol groups (with the latter process activated at lower temperatures than the former). Around ambient temperature a relaxation proposed to be the glass transition was observed and, in granular starch only, gelatinisation caused a relaxation around 60–80°C. No major differences were discernible between different starch types. The water content was the most important influence on the position of the relaxations, since water acted as a plasticiser.

Assembly Pathway of a Designed α-Helical Protein Fiber

Interest in the design of peptide-based fibrous materials is growing because it opens possibilities to explore fundamental aspects of peptide self-assembly and to exploit the resulting structures--for example, as scaffolds for tissue engineering. Here we investigate the assembly pathway of self-assembling fibers, a rationally designed alpha-helical coiled-coil system comprising two peptides that assemble on mixing. The dimensions spanned by the peptides and final structures (nanometers to micrometers), and the timescale over which folding and assembly occur (seconds to hours), necessitate a multi-technique approach employing spectroscopy, analytical ultracentrifugation, electron and light microscopy, and protein design to produce a physical model. We show that fibers form via a nucleation and growth mechanism. The two peptides combine rapidly (in less than seconds) to form sticky ended, partly helical heterodimers. A lag phase follows, on the order of tens of minutes, and is concentration-dependent. The critical nucleus comprises six to eight partially folded dimers. Growth is then linear in dimers, and subsequent fiber growth occurs in hours through both elongation and thickening. At later times (several hours), fibers grow predominantly through elongation. This kinetic, biomolecular description of the folding-and-assembly process allows the self-assembling fiber system to be manipulated and controlled, which we demonstrate through seeding experiments to obtain different distributions of fiber lengths. This study and the resulting mechanism we propose provide a potential route to achieving temporal control of functional fibers with future applications in biotechnology and nanoscale science and technology.