Flow-Induced Protein Chain Deformation, Segmental Orientation, and Phase Separation in Native Silk Feedstock.

The ability of many arthropods to spin silk and its many uses bear testament to its importance in Nature. Despite over a century of research, however, the spinning process is still not fully understood. While it is widely accepted that flow and chain alignment may be involved, the link to protein gelation remains obscure. Using combinations of rheology, polarized light imaging, and infrared spectroscopy to probe different length scales, this work explored flow-induced gelation of native silk feedstock from Bombyx mori larvae. Protein chain deformation, orientation, and microphase separation were observed, culminating in the formation of antiparallel ß-sheet structures while the work rate during flow appeared as an important criterion. Moreover, infrared spectroscopy provided direct observations suggesting a loss of protein hydration during flow-induced gelation of fibroin in native silk feedstock, which is consistent with recently reported hypotheses.

Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform.

Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to ß-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density.

Seeking Solvation: Exploring the Role of Protein Hydration in Silk Gelation.

The mechanism by which arthropods (e.g., spiders and many insects) can produce silk fibres from an aqueous protein (fibroin) solution has remained elusive, despite much scientific investigation. In this work, we used several techniques to explore the role of a hydration shell bound to the fibroin in native silk feedstock (NSF) from Bombyx mori silkworms. Small angle X-ray and dynamic light scattering (SAXS and DLS) revealed a coil size (radius of gyration or hydrodynamic radius) around 12 nm, providing considerable scope for hydration. Aggregation in dilute aqueous solution was observed above 65 °C, matching the gelation temperature of more concentrated solutions and suggesting that the strength of interaction with the solvent (i.e., water) was the dominant factor. Infrared (IR) spectroscopy indicated decreasing hydration as the temperature was raised, with similar changes in hydration following gelation by freezing or heating. It was found that the solubility of fibroin in water or aqueous salt solutions could be described well by a relatively simple thermodynamic model for the stability of the protein hydration shell, which suggests that the affected water is enthalpically favoured but entropically penalised, due to its reduced (vibrational or translational) dynamics. Moreover, while the majority of this investigation used fibroin from B. mori, comparisons with published work on silk proteins from other silkworms and spiders, globular proteins and peptide model systems suggest that our findings may be of much wider significance.

Magnesium Aluminium Silicate-Metformin Hydrochloride Complexes - The Use of Isothermal Calorimetry for Probing Clay and Drug Nanocomplexations.

BACKGROUND: Studying complexation between a wide variety of drugs and clay is of high importance in expanding the knowledge about controlled drug delivery and its exploitation. This study reports the use of isothermal calorimetry (ITC) in understanding the complexation process occurring between magnesium aluminium silicate (MAS) and metformin hydrochloride (MET), as a potentially controlled release drug delivery system. OBJECTIVES: To fully characterise and understand the complexes formed between MAS and MET and how that might impact on controlled release systems. METHODS: MAS and MET complex dispersions and particles were formulated and analysed using ITC, DSC, XRPD, ATR-FTIR, SEM/EDX, digital microscopy and 2D-SAXS. RESULTS: The calorimetric results confirmed the binding between MET and MAS at various pHs (5, 7 and 9) and temperatures (25 ºC and 37 ºC). The overall change in enthalpy was found to be exothermic with a comparatively small entropic contribution to the total change in Gibbs free energy, implying that the binding was an enthalpically driven process. These findings suggest that the binding process was dominated by hydrogen bonding and electrostatic interactions. pH and temperature variation did not have a great impact on the binding, as observed from the similarity in enthalpy (ΔH), entropy (ΔS) or Gibbs free energy (ΔG), with the reaction being only slightly more exothermic at pH 5 and at 37 ºC. 2D-SAXS was able to differentiate between MAS particulates and MAS-MET complexes when analysed in their liquid form suggesting the importance of appropriate methodology and instrumentation used in characterisation. CONCLUSION: ITC was successfully used in understanding the complexation process occurring between MAS and MET. Care and consideration however should thus be taken in the accurate determination and characterisation techniques for the formation of complexes for controlled release using MAS.

Stretching of Bombyx mori Silk Protein in Flow.

The flow-induced self-assembly of entangled Bombyx mori silk proteins is hypothesised to be aided by the 'registration' of aligned protein chains using intermolecularly interacting 'sticky' patches. This suggests that upon chain alignment, a hierarchical network forms that collectively stretches and induces nucleation in a precisely controlled way. Through the lens of polymer physics, we argue that if all chains would stretch to a similar extent, a clear correlation length of the stickers in the direction of the flow emerges, which may indeed favour such a registration effect. Through simulations in both extensional flow and shear, we show that there is, on the other hand, a very broad distribution of protein-chain stretch, which suggests the registration of proteins is not directly coupled to the applied strain, but may be a slow statistical process. This qualitative prediction seems to be consistent with the large strains (i.e., at long time scales) required to induce gelation in our rheological measurements under constant shear. We discuss our perspective of how the flow-induced self-assembly of silk may be addressed by new experiments and model development.

Native-like Flow Properties of an Artificial Spider Silk Dope.

Recombinant spider silk has emerged as a biomaterial that can circumvent problems associated with synthetic and naturally derived polymers, while still fulfilling the potential of the native material. The artificial spider silk protein NT2RepCT can be produced and spun into fibers without the use of harsh chemicals and here we evaluate key properties of NT2RepCT dope at native-like concentrations. We show that NT2RepCT recapitulates not only the overall secondary structure content of a native silk dope but also emulates its viscoelastic rheological properties. We propose that these properties are key to biomimetic spinning and that optimization of rheological properties could facilitate successful spinning of artificial dopes into fibers.

The influence of metal ions on native silk rheology.

Whilst flow is the basis for silk fibre formation, subtle changes in a silk feedstocks' chemical environment may serve to increase both energetic efficiency and control hierarchical structure development during spinning. Despite the role of pH being largely understood, the influence of metal ions is not, only being inferred by correlative work and observations. Through a combination of rheology and microscopy, we provide a causative study of how the most abundant metal ions in the silk feedstock, Ca2+ and K+, affect its flow properties and structure. Our results show that Ca2+ ions increase viscosity and prevent molecular alignment and aggregation, providing ideal storage conditions for unspun silk. In contrast, the addition of K+ ions promotes molecular alignment and aggregation and therefore seems to transfer the silk feedstock into a spinning state which confirms recent 'sticky reptation' modelling hypotheses. Additionally, we characterised the influence of the ubiquitous kosmotropic agent Li+, used to prepare regenerated silk solutions, and find that it promotes molecular alignment and prevents aggregation which may permit a range of interesting artificial silk processing techniques to be developed. In summary, our results provide a clearer picture of how metal ions co-ordinate, control and thus contribute towards silk protein self-assembly which in turn can inspire structuring approaches in other biopolymer systems.

Silk Protein Solution: A Natural Example of Sticky Reptation.

Silk is one of the most intriguing examples of biomolecular self-assembly, yet little is understood of molecular mechanisms behind the flow behavior generating these complex high-performance fibers. This work applies the polymer physics of entangled solution rheology to present a first microphysical understanding of silk in the linear viscoelastic regime. We show that silk solutions can be approximated as reptating polymers with "sticky" calcium bridges whose strength can be controlled through the potassium concentration. This approach provides a new window into critical microstructural parameters, in particular identifying the mechanism by which potassium and calcium ions are recruited as a powerful viscosity control in silk. Our model constitutes a viable starting point to understand not only the "flow-induced self-assembly" of silk fibers but also a broader range of phenomena in the emergent field of material-focused synthetic biology.

Thermodynamics of clay-drug complex dispersions: Isothermal titration calorimetry and high-performance liquid chromatography.

An understanding of the thermodynamics of the complexation process utilized in sustaining drug release in clay matrices is of great importance. Several characterisation techniques as well as isothermal calorimetry were utilized in investigating the adsorption process of a model cationic drug (diltiazem hydrochloride, DIL) onto a pharmaceutical clay system (magnesium aluminium silicate, MAS). X-ray powder diffraction (XRPD), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and optical microscopy confirmed the successful formation of the DIL-MAS complexes. Drug quantification from the complexes demonstrated variable behaviour in the differing media used with DIL degrading to desacetyl diltiazem hydrochloride (DC-DIL) in the 2 M HCl media. Here also, the authors report for the first time two binding processes that occurred for DIL and MAS. A competitor binding model was thus proposed and the thermodynamics obtained suggested their binding processes to be enthalpy driven and entropically unfavourable. This information is of great importance for a formulator as care and consideration should be given with appropriate media selection as well as the nature of binding in complexes.

Changes in Silk Feedstock Rheology during Cocoon Construction: The Role of Calcium and Potassium Ions.

Variation in silk feedstocks is a barrier both to our understanding of natural spinning and biomimetic endeavors. To address this, compositional changes are investigated in feedstock specimens from the domesticated silkworm (Bombyx mori). It is found that the feedstock viscosity decreased systematically by over two orders of magnitude during cocoon construction. Potential factors such as protein concentration, molecular weight, pH, or the presence of trehalose are excluded, whereas a clear correlation appear between viscosity and the relative concentrations of Ca2+ and K+ ions. It is expected that Ca2+ ions would favor "salt bridges" between acidic (Asp and Glu) amino acids, leading to an increased viscosity, whereas K+ ions would compete for these sites, thereby reducing viscosity. Thus, these findings suggest a simple, systematic yet sophisticated control of feedstock viscosity in the silkworm, which in turn can be applied to future industrial silk production.

Extensional flow behaviour and spinnability of native silk.

Silk fibres are assembled via flow. While changes in the physiological environment of the gland as well as the shear rheology of silk are largely understood, the effect of extensional flow fields on native silk proteins is almost completely unknown. Here we demonstrate that filament stretching on a conventional tensile tester is a suitable technique to assess silk's extensional flow properties and its ability to form fibres under extensional conditions characteristic of natural spinning. We report that native Bombyx mori silk responds differently to extensional flow fields when compared to synthetic linear polymers, as evidenced by a higher Trouton ratio which we attribute to silk's increased interchain interactions. Finally, we show that native silk proteins can only be spun into stable fibres at low extension rates as a result of dehydration, suggesting that extensional fields alone are unable to induce natural fibre formation.

The Rheology behind Stress-Induced Solidification in Native Silk Feedstocks.

The mechanism by which native silk feedstocks are converted to solid fibres in nature has attracted much interest. To address this question, the present work used rheology to investigate the gelation of Bombyx mori native silk feedstock. Exceeding a critical shear stress appeared to be more important than shear rate, during flow-induced initiation. Compositional changes (salts, pH etc.,) were not required, although their possible role in vivo is not excluded. Moreover, after successful initiation, gel strength continued to increase over a considerable time under effectively quiescent conditions, without requiring further application of the initial stimulus. Gelation by elevated temperature or freezing was also observed. Prior to gelation, literature suggests that silk protein adopts a random coil configuration, which argued against the conventional explanation of gelation, based on hydrophilic and hydrophobic interactions. Instead, a new hypothesis is presented, based on entropically-driven loss of hydration, which appears to explain the apparently diverse methods by which silk feedstocks can be gelled.

Native Silk Feedstock as a Model Biopolymer: A Rheological Perspective.

Variability in silk's rheology is often regarded as an impediment to understanding or successfully copying the natural spinning process. We have previously reported such variability in unspun native silk extracted straight from the gland of the domesticated silkworm Bombyx mori and discounted classical explanations such as differences in molecular weight and concentration. We now report that variability in oscillatory measurements can be reduced onto a simple master-curve through normalizing with respect to the crossover. This remarkable result suggests that differences between silk feedstocks are rheologically simple and not as complex as originally thought. By comparison, solutions of poly(ethylene-oxide) and hydroxypropyl-methyl-cellulose showed similar normalization behavior; however, the resulting curves were broader than for silk, suggesting greater polydispersity in the (semi)synthetic materials. Thus, we conclude Nature may in fact produce polymer feedstocks that are more consistent than typical man-made counterparts as a model for future rheological investigations.

The Borrelia afzelii outer membrane protein BAPKO_0422 binds human factor-H and is predicted to form a membrane-spanning ß-barrel.

The deep evolutionary history of the Spirochetes places their branch point early in the evolution of the diderms, before the divergence of the present day Proteobacteria. As a spirochete, the morphology of the Borrelia cell envelope shares characteristics of both Gram-positive and Gram-negative bacteria. A thin layer of peptidoglycan, tightly associated with the cytoplasmic membrane, is surrounded by a more labile outer membrane (OM). This OM is rich in lipoproteins but with few known integral membrane proteins. The outer membrane protein A (OmpA) domain is an eight-stranded membrane-spanning ß-barrel, highly conserved among the Proteobacteria but so far unknown in the Spirochetes. In the present work, we describe the identification of four novel OmpA-like ß-barrels from Borrelia afzelii, the most common cause of erythema migrans (EM) rash in Europe. Structural characterization of one these proteins (BAPKO_0422) by SAXS and CD indicate a compact globular structure rich in ß-strand consistent with a monomeric ß-barrel. Ab initio molecular envelopes calculated from the scattering profile are consistent with homology models and demonstrate that BAPKO_0422 adopts a peanut shape with dimensions 25×45 Å (1 Å=0.1 nm). Deviations from the standard C-terminal signature sequence are apparent; in particular the C-terminal phenylalanine residue commonly found in Proteobacterial OM proteins is replaced by isoleucine/leucine or asparagine. BAPKO_0422 is demonstrated to bind human factor H (fH) and therefore may contribute to immune evasion by inhibition of the complement response. Encoded by chromosomal genes, these proteins are highly conserved between Borrelia subspecies and may be of diagnostic or therapeutic value.

Formulation and evaluation of floating mucoadhesive alginate beads for targeting Helicobacter pylori.

OBJECTIVES: There are various obstacles in the eradication of Helicobacter pylori infections, including low antibiotic levels and poor accessibility of the drug at the site of the infection. This study describes the preparation and characterisation of novel floating mucoadhesive alginate beads loaded with clarithromycin for delivery to the gastric mucosa to improve the eradication of this microorganism. METHODS: Calcium alginate beads were prepared by ionotropic gelation. The formulation was modified through addition of oil and coating with chitosan to improve floating, mucoadhesion and modify drug release. KEY FINDINGS: Scanning electron microscopy confirmed the sphericity of the beads with X-ray microtomography showing the three-dimensional structure of the beads with the layered internal structure of the bead and the even distribution of the drug within the bead. This formulation combined two gastro-retentive strategies, and produced excellent in-vitro floating, mucoadhesive and drug release characteristics. Enhanced stability of the beads in phosphate buffer raises a potential for the modified formulations to be targeted to regions of higher pH within the gastrointestinal tract. Drug release from these beads was sustained through an unstirred mucin layer simulating in-vivo conditions under which the H. pylori resides in the gastric mucosa. CONCLUSIONS: This novel formulation will ensure retention for a longer period in the stomach than conventional formulations and control drug release, ensuring high local drug concentrations, leading to improved eradication of the bacteria.

The influence of salt formation on electrostatic and compression properties of flurbiprofen salts.

Salt formation is an effective method of improving physicochemical properties of acidic and basic drugs. The selection of a salt form most suitable for drug development requires a well-designed screening strategy to ensure various issues are addressed in the early development stages. Triboelectrification of pharmaceutical powders may cause problems during processing such as segregation of components due to the effects of particle adhesion. However, very little work has been done on the effect of salt formation on triboelectrification properties. In this paper, salts of flurbiprofen were prepared by combining the drug with a selection of closely related amine counter ions. The aim of the work was to investigate the impact of the counter ion on electrostatic charge of the resultant salts to inform the salt selection process. The experimental results show the magnitude of charge and polarity of the flurbiprofen salts to be highly dependent on the type of counter ion selected for the salt formation. Furthermore, particle adhesion to the stainless steel surface of the shaking container and the salts' compression properties were measured. The formed salts had lower electrostatic charges, improved tabletability, and resulted in reduced adhesion of these powders compared with the parent drug.

The influence of agitation sequence and ionic strength on in vitro drug release from hypromellose (E4M and K4M) ER matrices--the use of the USP III apparatus.

Theophylline extended release (ER) matrices containing hypromellose (hydroxypropyl methylcellulose (HPMC) E4M and K4M were evaluated in media with a pH range of 1.2-7.5, using an automated USP type III, Bio-Dis dissolution apparatus. The objectives of this study were to evaluate the effects of systematic agitation, ionic strength and pH on the release of theophylline from the gel forming hydrophilic polymeric matrices with different methoxyl substitution levels. Tribo-electric charging of hypromellose, theophylline and their formulated blends containing E4M and K4M grades has been characterised, along with quantitative observations of flow, compression behaviour and particle morphology. Agitations were studied at 5, 10, 15, 20, 25, 30 dips per minute (dpm) and also in the ascending and descending order in the dissolution vials. The ionic concentration strength of the media was also varied over a range of 0-0.4M to simulate the gastrointestinal fed and fasted states and various physiological pH conditions. To study the effect of ionic strength on the hydrophilic matrices, agitation was set at 20 dpm. The charge results on individual components imply that the positively charged particles have coupled with the negatively charged particles to form a stable ordered mixture which is believed to result in a more homogeneous and stable system. The particle shape analysis showed the HPMC K4M polymer to have a more irregular morphology and a rougher surface texture in comparison to the HPMC E4M polymer, possibly a contributory factor to the gelation process. The results showed gelation occurred quicker for the K4M tablet matrices. Drug release increased with increased agitation. This was more pronounced for the E4M tablet matrices. The ionic strength also had more of an effect on the drug release from the E4M matrices. The experiments highlighted the resilience of the K4M matrices in comparison with the E4M matrices. The results thus show that despite similar viscosities of E4M and K4M, the methoxyl substitution makes a difference to their control of drug release and as such care and consideration should be given to the choice of polymer used for extended release. The use of systematic change of agitation method and ionic strength may indicate potential fed and fasted effects on drug release from hydrophilic matrices.

Variations in compaction behaviour for tablets of different size and shape, revealed by small-angle X-ray scattering.

Local variations in compaction behaviour were investigated, for specimens of different shapes and thickness, comparing predictions from finite element (FE) modelling and results from a recently developed method using small-angle X-ray scattering (SAXS). Good agreement was generally obtained between these methods, in terms of the variations of density, compaction strain and principal strain direction within the specimens examined. The combination of SAXS and FE methods appeared particularly suitable for studying pharmaceutical tablets, revealing effects (such as nano-strain of intragranular morphology and strain direction) that are not easily observed by other methods, and which may have significant effects on tablet integrity or swelling and drug delivery characteristics.

Examination of hard segment and soft segment phase separation in polyurethane medical materials by electron microscopy techniques.

A combination of transmission electron microscopy (TEM) and in situ tensile testing in an environmental scanning electron microscopy (ESEM) was used to evaluate the static bulk and dynamic surface morphologies of medical polyurethanes. TEM results showed phase-separated hard segment and soft segment structures. Surface morphology as a function of strain was studied using ESEM in conjunction with a tensometer.

Effect of processing route and acetone pre-treatment on the biostability of pellethane materials used in medical device applications.

Thermoplastic polyurethanes, such as Pellethane 2363 80A (Pel80A) and Pellethane 2363 55D (Pel55D) are widely used in the medical device industry because of their biological and mechanical properties. However, premature failure in such devices has been observed and attributed to environmental stress cracking (ESC). The current work investigates the possibility of reducing ESC via bulk morphology manipulation. This can be achieved through various processing routes such as solvent-casting (SC) and hot-press quenching (HPQ). The effect of stress on the bulk morphology of Pel55D and Pel80A was evaluated using small-angle X-ray scattering (SAXS) in conjunction with tensile testing. SC samples exhibited greater phase separation compared with HPQ samples. Alignment of hard segment domains became apparent around the point of yield. Onset of ESC with respect to SC and HPQ routines was determined using the Zhao-Stokes glass-wool test with optical (OM) and environment scanning electron microscopy (ESEM). Improvement in biostability of Pel80A was found in HPQ samples compared to those that were SC. A secondary objective of this work was to investigate the effect of acetone pre-treatment on surface morphology. High resolution imaging of acetone treated and untreated SC Pel80A showed significant differences in surface morphology.