Peggy Cebe

Crystallization behaviour of poly(ether-ether-ketone)

Abstract A study has been made of the crystallization behaviour of poly(ether-ether-ketone), PEEK, under isothermal and non-isothermal conditions. A differential scanning calorimeter was used to monitor the energetics of the crystallization process from the melt and from the rubbery amorphous state. During isothermal crystallization, relative crystallinity develops with a time dependence described by the Avrami equation, with exponent n = 3. Greater absolute crystallinity develops during melt crystallization, nearly twice that which develops from the rubbery state, for comparable rates of crystallization. For non-isothermal studies, amorphous films were crystallized by heating or cooling at rates from 1°C/min to 50°C/min. A large fraction of crystallinity, from 0.45 to 0.70, develops by secondary processes. A kinetic treatment based on the Avrami equation is presented to describe the primary processes leading to non-isothermal crystallization. We have calculated activation energies of 68 kcal mol−1 for crystallization from the melt, and 52 kcal mol−1 for crystallization from the rubbery amorphous state. Results of isothermal and non-isothermal crystallization of PEEK are compared with those of poly(ethylene terephthalate).

Water-Insoluble Silk Films with Silk I Structure

Water-insoluble regenerated silk materials are normally produced by increasing the beta-sheet content (silk II). In the present study water-insoluble silk films were prepared by controlling the very slow drying of Bombyx mori silk solutions, resulting in the formation of stable films with a predominant silk I instead of silk II structure. Wide angle X-ray scattering indicated that the silk films stabilized by slow drying were mainly composed of silk I rather than silk II, while water- and methanol-annealed silk films had a higher silk II content. The silk films prepared by slow drying had a globule-like structure at the core surrounded by nano-filaments. The core region was composed of silk I and silk II, surrounded by hydrophilic nano-filaments containing random turns and alpha-helix secondary structures. The insoluble silk films prepared by slow drying had unique thermal, mechanical and degradative properties. Differential scanning calorimetry results revealed that silk I crystals had stable thermal properties up to 250 degrees C, without crystallization above the T(g), but degraded at lower temperatures than silk II structure. Compared with water- and methanol-annealed films the films prepared by slow drying had better mechanical ductility and were more rapidly enzymatically degraded, reflecting the differences in secondary structure achieved via differences in post processing of the cast silk films. Importantly, the silk I structure, a key intermediate secondary structure for the formation of mechanically robust natural silk fibers, was successfully generated by the present approach of very slow drying, mimicking the natural process. The results also point to a new mode of generating new types of silk biomaterials with enhanced mechanical properties and increased degradation rates, while maintaining water insolubility, along with a low beta-sheet content.

Regulation of Silk Material Structure by Temperature-Controlled Water Vapor Annealing

We present a simple and effective method to obtain refined control of the molecular structure of silk biomaterials through physical temperature-controlled water vapor annealing (TCWVA). The silk materials can be prepared with control of crystallinity, from a low content using conditions at 4 °C (α helix dominated silk I structure), to highest content of ∼60% crystallinity at 100 °C (β-sheet dominated silk II structure). This new physical approach covers the range of structures previously reported to govern crystallization during the fabrication of silk materials, yet offers a simpler, green chemistry, approach with tight control of reproducibility. The transition kinetics, thermal, mechanical, and biodegradation properties of the silk films prepared at different temperatures were investigated and compared by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), uniaxial tensile studies, and enzymatic degradation studies. The results revealed that this new physical processing method accurately controls structure, in turn providing control of mechanical properties, thermal stability, enzyme degradation rate, and human mesenchymal stem cell interactions. The mechanistic basis for the control is through the temperature-controlled regulation of water vapor to control crystallization. Control of silk structure via TCWVA represents a significant improvement in the fabrication of silk-based biomaterials, where control of structure-property relationships is key to regulating material properties. This new approach to control crystallization also provides an entirely new green approach, avoiding common methods that use organic solvents (methanol, ethanol) or organic acids. The method described here for silk proteins would also be universal for many other structural proteins (and likely other biopolymers), where water controls chain interactions related to material properties.

Vortex-induced injectable silk fibroin hydrogels.

A novel, to our knowledge, technique was developed to control the rate of beta-sheet formation and resulting hydrogelation kinetics of aqueous, native silk solutions. Circular dichroism spectroscopy indicated that vortexing aqueous solutions of silkworm silk lead to a transition from an overall protein structure that is initially rich in random coil to one that is rich in beta-sheet content. Dynamic oscillatory rheology experiments collected under the same assembly conditions as the circular dichroism experiments indicated that the increase in beta-sheet content due to intramolecular conformational changes and intermolecular self-assembly of the silk fibroin was directly correlated with the subsequent changes in viscoelastic properties due to hydrogelation. Vortexing low-viscosity silk solutions lead to orders-of-magnitude increase in the complex shear modulus, G*, and formation of rigid hydrogels (G* approximately 70 kPa for 5.2 wt % protein concentration). Vortex-induced, beta-sheet-rich silk hydrogels consisted of permanent, physical, intermolecular crosslinks. The hydrogelation kinetics could be controlled easily (from minutes to hours) by changing the vortex time, assembly temperature and/or protein concentration, providing a useful timeframe for cell encapsulation. The stiffness of preformed hydrogels recovered quickly, immediately after injection through a needle, enabling the potential use of these systems for injectable cell delivery scaffolds.

Mechanism of enzymatic degradation of beta-sheet crystals

The anti-parallel beta pleated sheet is a fundamental secondary structure in proteins and a major component in silk fibers generated by silkworms and spiders, with a key role to stabilize these proteins via physical cross-links. Importantly, these beta-sheets are fully degradable and nontoxic structures in biology, in contrast for example to beta-amyloid structures formed in disease states. Thus, insight into mechanism of enzymatic degradation would be instructive as a route to elucidating differences among these stable yet different structural features in biological systems. We report on the mechanism of enzymatic degradation of anti-parallel beta pleated sheets with Bombyx mori silk structures, leading to fibrils and subsequently to nanofilaments (2nm thickness and 160nm length). These nanofilaments play a role as nucleators of the crystalline regions, an important feature of the system that can be exploited to design silk-based biomaterials with predictable biodegradability and mechanical properties. The potential toxicity of degradation products from these proteolytic enzymes was also assessed in vitro and no cell toxicity found in vitro for the protease found in vivo in the human body. The degradation mechanism of beta-sheet silk crystals provides additional insight into the significant differences in biological impact between the anti-parallel beta-sheet silk biomaterials reported in this work vs. amyloid structures in disease states, adding to prior descriptions of chemical and structural differences that are more extensively documented.

Protein based Block Copolymers

Advances in genetic engineering have led to the synthesis of protein-based block copolymers with control of chemistry and molecular weight, resulting in unique physical and biological properties. The benefits from incorporating peptide blocks into copolymer designs arise from the fundamental properties of proteins to adopt ordered conformations and to undergo self-assembly, providing control over structure formation at various length scales when compared to conventional block copolymers. This review covers the synthesis, structure, assembly, properties, and applications of protein-based block copolymers.

Protein-based composite materials

Protein-based composite biomaterials have been actively pursued as they can encompass a range of physical properties to accommodate a broader spectrum of functional requirements, such as elasticity to support diverse tissues. By optimizing molecular interfaces between structural proteins, useful composite materials can be fabricated as films, gels, particles, and fibers, as well as for electrical and optical devices. Such systems provide analogies to more traditional synthetic polymers yet with expanded utility due to the materials tunability, mechanical properties, degradability, biocompatibility, and functionalization, such as for drug delivery, biosensors, and tissue regeneration.

Self-Assembly of Genetically Engineered Spider Silk Block Copolymers

The design, construction, and preliminary characterization of a novel family of spider silk-like block copolymers are described. The design was based on the assembly of individual spider silk modules, in particular, polyalanine (A) and glycine-rich (B) blocks, that display different phase behavior in aqueous solution. Spider silk was chosen as a model for these block copolymer studies based on its extraordinary material properties, such as toughness, biocompatibility, and biodegradability. Trends in spider silk-like block copolymer secondary structure and assembly behavior into specific material morphologies were determined as a function of the number of hydrophobic blocks, the presence of a hydrophilic purification tag and solvent effects. Structures and morphologies were assessed by Fourier transform infrared spectroscopy and scanning electron microscopy. In terms of structure, beta-sheet content increased with an increase in the number of polyalanine blocks, and the purification tag had significant impact on the secondary structure. In terms of morphology, spheres, rod-like structures, bowl-shaped micelles, and giant compound micelles were observed and the morphologies were linked with the size of the hydrophobic block, the presence of the purification tag, and the solvent environment. This study provides a basis for future designs of smart biomaterials based on spider silk chemistries, with controlled structure-architecture-function relationships.

Melting behaviour of poly (phenylene sulphide): 2. Multiple stage melt crystallization

Abstract We have studied the melting behaviour of poly (phenylene sulphide), PPS, that has been crystallized from the melt over a wide range of undercooling conditions. Two grades of PPS were used in the study: low molecular weight Ryton V-1 and an experimental medium molecular weight film grade. In this work we report the melting behaviour after a single stage of isothermal melt crystallization. In nearly all cases, dual or triple melting endotherms are seen, but the location and shape of the uppermost endotherm depend upon the degree of undercooling from the melt and on the prior thermal history. At a low degree of undercooling, for both materials, dual endotherms are observed and both melting points increase with the crystallization temperature. Using the immediate rescan technique, we show that the dual endotherms exist together very early in the crystallization process, when only a small fraction of crystals have formed. These results suggest that multiple crystal perfections form early on in the crystallization at low undercooling. As the degree of undercooling increases, the melting point of the uppermost endotherm becomes independent of the crystallization temperature. This result, and the appearance of a triple endothermic response for Ryton at the highest undercooling, indicate that now reorganization of imperfect crystals is dominating the observed endothermic response. We present a model in which the distribution of crystal perfections created during melt crystallization controls the multiple melting behaviour of PPS. At low undercooling conditions, a bimodal distribution of crystals can form which eventually may become two morphologies. At high undercooling conditions, a broad distribution of crystals can form, a part of which may melt and reorganize during the normal d.s.c. scan.

Stabilization and release of enzymes from silk films.

A significant challenge remains to protect protein drugs from inactivation during production, storage, and use. In the present study, the stabilization and release of horseradish peroxidase (HRP) in silk films was investigated. Water-insoluble silk films were prepared under mild aqueous conditions, maintaining the activity of the entrapped enzyme. Depending on film processing and post-processing conditions, HRP retained more than 90% of the initial activity at 4 degrees C, room temperature and 37 degrees C over two months. The stability of protein drugs in silk films is attributed to intermolecular interactions between the silk and the enzymes, based on Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The unique structural feature of silk molecules, periodic hydrophobic-hydrophilic domains, enabled strong interactions with proteins. The entrapped protein was present in two states, untrapped active and trapped inactive forms. The ratio between the two forms varied according to processing conditions. Proteolytic degradation and dissolution of the silk films resulted in the release of the bound enzyme which was otherwise not released by diffusion; enzyme recovered full activity upon release. There was a linear relationship between silk degradation/dissolution and the release of entrapped enzyme. Modifying the secondary structure of the silk matrix and the interactions with the non-crystalline domains resulted in control of the film degradation or dissolution rate, and therefore the release rate of the entrapped enzyme. Based on the above results, silk materials are an intriguing carrier for proteins in terms of both retention of activity and controllable release kinetics from the films.