Molecular weight distribution and solution properties of silk fibroins with different dissolution conditions

Four regenerated silk fibroin (SF) samples were prepared under different dissolution conditions and their molecular weight (MW) distributions and solution properties in water and formic acid were examined. SFL, produced by dissolving in LiBr aqueous solution for 6h, showed the highest MW level. In the three SFC samples, produced by dissolving SF in CaCl(2)/H(2)O/EtOH solution for dissolution times ranging from 3 to 180 min, the MW of the SFs decreased with increasing dissolution time and a new band appeared at low MW. Interestingly, SFL presented as a relatively transparent aqueous solution with 10-30 nm particle size, whereas the three SFC samples exhibited a turbid solution with 100-300 nm particle size. SF formic acid solutions showed a higher viscosity than SF aqueous solutions and exhibited almost Newtonian fluid behavior, whereas SF aqueous solutions displayed abrupt shear thinning in the low shear rate region (0.1-3 s(-1)).     

Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid

Structural characteristics and thermal and solution properties of the regenerated silk fibroin (SF) prepared from formic acid (FU) were compared with those of SF from water (AU). According to the turbidity and shear viscosity measurement, SF formic acid solution was stable and transparent, no molecular aggregations occurred. The sample FU exhibited the beta-sheet structure, while AU random coil conformation using Fourier transform infrared (FTIR), X-ray diffraction (XRD), and differential scanning calorimetry. The effects of methanol treatment on samples were also examined. According to the measurement of crystallinity (XRD) and crystallinity index (FTIR), the concept of long/short-range ordered structure formation was proposed. Long-range ordered crystallites are predominantly formed for methanol treated SF film while SF film cast from formic acid favors the formation of short-range ordered structure. The relaxation temperatures of SF films measured by dynamic thermomechanical analysis supported the above mechanism due to the sensitivity of relaxation temperature on the short-range order.     

Enhanced Dissolution and Stability of Lansoprazole by Cyclodextrin Inclusion Complexation: Preparation, Characterization, and Molecular Modeling

In this study, lansoprazole (LSP)/cyclodextrin (CD) inclusion complexes were prepared using a fluid bed coating technique, with β-cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (HPCD) as the host molecules, respectively, to simultaneously improve the dissolution and stability of LSP. The dissolution rate and stability of LSP was dramatically enhanced by inclusion complexation regardless of CD type. LSP/HPCD inclusion complex was more stable under illumination than LSP/β-CD inclusion complex. Differential scanning calorimetry and powder X-ray diffractometry proved the absence of crystallinity in both LSP/CD inclusion complexes. Fourier transform infrared spectroscopy together with molecular modeling indicated that the benzimidazole of LSP was included in the cavity of both CDs, while LSP was more deeply included in HPCD than β-CD. The enhanced photostability was due to the inclusion of the sulfinyl moiety into the HPCD cavity. CD inclusion complexation could improve the dissolution and stability of LSP.KEY WORDS: cyclodextrin, dissolution, inclusion complex, lansoprazole, molecular modeling, stability     

Biodegradable materials based on silk fibroin and keratin

Wool and silk were dissolved and used for the preparation of blended films. Two systems are proposed: (1) blend films of silk fibroin and keratin aqueous solutions and (2) silk fibroin and keratin dissolved in formic acid. The FTIR spectra of pure films cast from aqueous solutions indicated that the keratin secondary structure mainly consists of alpha-helix and random coil conformations. The IR spectrum of pure SF is characteristic of films with prevalently amorphous structure (random coil conformation). Pure keratin film cast from formic acid shows an increase in the amount of beta-sheet and disordered keratin structures. The FTIR pattern of SF dissolved in formic acid is characteristic of films with prevalently beta-sheet conformations with beta-sheet crystallites embedded in an amorphous matrix. The thermal behavior of the blends confirmed the FTIR results. DSC curve of pure SF is typical of amorphous SF and the curve of pure keratin show the characteristic melting peak of alpha-helices for the aqueous system. These patterns are no longer observed in the films cast from formic acid due to the ability of formic acid to induce crystallization of SF and to increase the amount of beta-sheet structures on keratin. The nonlinear trend of the different parameters obtained from FTIR analysis and DSC curves of both SF/keratin systems indicate that when proteins are mixed they do not follow additives rules but are able to establish intermolecular interactions. Degradable polymeric biomaterials are preferred candidates for medical applications. It was investigated the degradation behavior of both SF/keratin systems by in vitro enzymatic incubation with trypsin. The SF/keratin films cast from water underwent a slower biological degradation than the films cast from formic acid. The weight loss obtained is a function of the amount of keratin in the blend. This study encourages the further investigation of the type of matrices presented here to be applied whether in scaffolds for tissue engineering or as controlled release drug delivery vehicles.     

Formation of silk fibroin matrices with different texture and its cellular response to normal human keratinocytes

Three forms of silk fibroin (SF) matrices, woven (microfiber), non-woven (nanofiber), and film form, were used to perform a conformational analysis and cell culture using normal human oral keratinocytes (NHOK). To obtain the SF microfiber (SF-M) matrix, natural grey silk was degummed, while the SF film (SF-F) and nanofiber (SF-N) matrices were prepared by casting and electrospinning the formic acid solutions of the regenerated SF, respectively. For insolubilization, as-prepared SF-F and SF-N matrices were chemically treated with an aqueous methanol solution of 50%. The conformational structures of as-prepared and chemically treated SF matrices were investigated using attenuated total reflectance infrared spectroscopy (ATR-IR) and solid-state ¹³C CP/MAS nuclear magnetic resonance (NMR) spectroscopy. The as-cast SF-F matrix formed a mainly β-sheet structure that was similar to the SF-M matrix, whereas the as-spun SF-N matrix had a random coil conformation as the predominant secondary structure. Conformational transitions from random coil to β-sheet of the as-spun SF-N occurred rapidly within 10 min following aqueous methanol treatment, and were confirmed by solid-state ¹³C NMR analysis. To assess the cytocompatibility and cells behavior on the different textures of SF, we examined the cell attachment and spreading of NHOK that was seeded onto the SF matrices, as well as the interaction between the cells and SF matrices. Our results indicate that the SF nanofiber matrix may be more preferable than SF film and SF microfiber matrices for biomedical applications, such as wound dressings and scaffolds for tissue engineering.     

Formation of Itraconazole–Succinic Acid Cocrystals by Gas Antisolvent Cocrystallization

Cocrystals of itraconazole, an antifungal drug with poor bioavailability, and succinic acid, a water-soluble dicarboxylic acid, were formed by gas antisolvent (GAS) cocrystallization using pressurized CO₂ to improve itraconazole dissolution. In this study, itraconazole and succinic acid were simultaneously dissolved in a liquid solvent, tetrahydrofuran, at ambient conditions. The solution was then pressurized with CO₂, which decreased the solvating power of tetrahydrofuran and caused crystallization of itraconazole–succinic acid cocrystals. The cocrystals prepared by GAS cocrystallization were compared to those produced using a traditional liquid antisolvent, n-heptane, for crystallinity, chemical structure, thermal behavior, size and surface morphology, potential clinical relevance, and stability. Powder X-ray diffraction, Fourier transform infrared spectroscopy, differential scanning calorimetry, and scanning electron microscopy analyses showed that itraconazole–succinic acid cocrystals with physical and chemical properties similar to cocrystals produced using a traditional liquid antisolvent technique can be prepared by CO₂ antisolvent cocrystallization. The dissolution profile of itraconazole was significantly enhanced through GAS cocrystallization with succinic acid, achieving over 90% dissolution in less than 2 h. The cocrystals appeared stable against thermal stress for up to 4 weeks under accelerated stability conditions, showing only moderate decreases in their degree of crystallinity but no change in their crystalline structure. This study shows the utility of an itraconazole–succinic acid cocrystal for improving itraconazole bioavailability while also demonstrating the potential for CO₂ to replace traditional liquid antisolvents in cocrystal preparation, thus making cocrystal production more environmentally benign and scale-up more feasible.KEY WORDS: cocrystals, dissolution rate, gas antisolvent, itraconazole     

Preparation and characterization of wet spun silk fibroin/poly(vinyl alcohol) blend filaments

Silk fibroin (SF)/poly(vinyl alcohol) (PVA) blend filaments were prepared by a wet spinning process. Regenerated SF and PVA were dissolved in formic acid and the dope solution exhibited good fiber formation in a methanol coagulation bath. Due to the miscibility of SF/PVA in formic acid, the filament had a smooth surface and dense structure with a circular cross-section. The crystalline structure and thermal properties were varied with different SF/PVA ratios. The mechanical properties of the filament were also controlled by blending PVA with SF. Especially, the knot strength of the SF filament, which is a very important suture property, could be significantly improved by blending with PVA.     

Perspective on Water of Crystallization Affecting the Functionality of Pharmaceuticals

Water can be incorporated into crystalline lattice of organic molecules in several ways and thus forms systems with different molecular packing characteristics. This review outlines a general classification of hydrates and explains why it is of high relevance for pharmaceutical researchers to investigate water of crystallization and hydrate systems. The different manufacturing steps related to the final drug product are also briefly discussed with an emphasis on the role of water and possible solid-state transformations related to hydrates. Application of spectroscopic techniques in characterizing water of crystallization and the effect of water on the drug and formulation stability are presented. Moreover, the role of water on the in vitro drug dissolution behavior is reviewed where an example is shown to illustrate the recent development on correlating solid state of the drug, for instance, hydrate formation, to the dissolution profile of the product, to achieve a better understanding of the dissolution process.     

Mixed protein blends composed of gelatin and Bombyx mori silk fibroin: effects of solvent-induced crystallization and composition

Novel protein blends have been prepared by mixing gelatin (G) with Bombyx mori silk fibroin (SF) and using aqueous methanol (MeOH) to post-induce SF crystallization. When co-cast from solution, amorphous blends of these polymers appear homogeneous, as discerned from visual observation, microscopy, and Fourier-transform infrared (FTIR) spectroscopy. Upon subsequent exposure to aqueous MeOH, SF undergoes a conformational change from random coil to beta sheet. This transformation occurs in pure SF, as well as in each of the G/SF blends, according to X-ray diffractometry and thermal calorimetry. The influence of MeOH-induced SF crystallization on structure and property development has been ascertained in terms of preparation history and blend composition. Thermal gravimetric analysis reveals that the presence of beta sheets in SF and G/SF blends improves thermal stability, while extensional rheometry confirms that SF crystallization enhances the tensile properties of the blends. By preserving a support scaffold above the G helix-to-coil transition temperature, the formation of crystalline SF networks in G/SF blends can be used to stabilize G-based hydrogels for biomaterial and pharmaceutical purposes. The present study not only examines the properties of G/SF blends before and after SF crystallization, but also establishes the foundation for future research into thermally responsive G/SF bioconjugates.     

Alpha-helix formation by solvent-solvent interaction

The degree of helicity θ of a series of homologous polypeptides as a function of solvent composition was investigated. The polypeptides studied were: poly-N⁵-(3-hydroxypropyl)-L -glutamine (PHPG) as well as the corresponding 2-hydroxyethyl and 4-hydoxybutyl derivatives (PHKG and PHBG, respectively). PHPG, which is nonhelical in formic acid, attains helicity on addition of relatively small amounts of formates, formamide, and urea to its solution in formic acid. This demonstrates that the high acidity of pure formic acid is largely responsible for its helix-breaking power-probably through protonation of the peptide bonds. In formic acid-water mixtures all three polymers show a maximum in degree of helicity at a mole fraction of about 0.3 formic acid. This is interpreted as being due to interaction between the two helix-breaking solvents, which results in the formation of an inactive molecular species. It is shown that solvent-induced transitions with helicity maxima are predicted by the Bixon-Lifson treatment when applied to this system.     

Kinetic and stoichiometric characterization of organoautotrophic growth of Ralstonia eutropha on formic acid in fed‐batch and continuous cultures

Formic acid, acting as both carbon and energy source, is a safe alternative to a carbon dioxide, hydrogen and dioxygen mix for studying the conversion of carbon through the Calvin–Benson–Bassham (CBB) cycle into value-added chemical compounds by non-photosynthetic microorganisms. In this work, organoautotrophic growth of Ralstonia eutropha on formic acid was studied using an approach combining stoichiometric modeling and controlled cultures in bioreactors. A strain deleted of its polyhydroxyalkanoate production pathway was used in order to carry out a physiological characterization. The maximal growth yield was determined at 0.16 Cmole Cmole⁻¹ in a formate-limited continuous culture. The measured yield corresponded to 76% to 85% of the theoretical yield (later confirmed in pH-controlled fed-batch cultures). The stoichiometric study highlighted the imbalance between carbon and energy provided by formic acid and explained the low growth yields measured. Fed-batch cultures were also used to determine the maximum specific growth rate (μ_max = 0.18 h⁻¹) and to study the impact of increasing formic acid concentrations on growth yields. High formic acid sensitivity was found in R eutropha since a linear decrease in the biomass yield with increasing residual formic acid concentrations was observed between 0 and 1.5 g l⁻¹.     

Structure,phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3′-splice site recognition

Recognition of the 3′-splice site is a key step in pre-mRNA splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein–protein and protein–RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1^NTD). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1^NTD with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65^UHM) reveals that, in addition to the known U2AF65^UHM–SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65^UHM, which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1–U2AF65–RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82-Pro83 motif rigidifies a long unstructured linker in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1–U2AF65 or the SF1–U2AF65–RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 3′-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1–U2AF65–RNA complex.     

Investigation of rheological properties and conformation of silk fibroin in the solution of AmimCl

The conformation and eventual morphology of silk fibroin (SF) chains are crucial for the mechanical properties of SF materials, and are strongly related to the solvation step as a key stage in their processing conditions. In this work, a novel SF/AmimCl (1-allyl-3-methylimidazolium chloride) solution with unique properties is reported and compared with conventional regenerated SF aqueous solutions, based on an investigation of its rheological properties. The steady shearing behavior suggested that AmimCl is a good solvent for SF molecules, and shear thinning of semidiluted SF/AmimCl solution at high shear rates showed behavior similar to that in native spinning, which is due to the rearrangement and orientation of SF molecular chains. Fitting of experimental dynamic viscoelastic data to the Rouse model provided an effective method to estimate the molecular weight of SF. We believe that this work not only provides a better understanding of the relationship between properties of silk protein and aggregation states of their molecular chains, but also provides tools to fabricate high-performance SF-based materials.     

Wetting Kinetics: an Alternative Approach Towards Understanding the Enhanced Dissolution Rate for Amorphous Solid Dispersion of a Poorly Soluble Drug

Developing amorphous solid dispersions of water-insoluble molecules using polymeric materials is a well-defined approach to improve the dissolution rate and bioavailability. While the selected polymer plays a vital role in stabilizing the amorphous solid dispersion physically, it is equally important to improve the dissolution profile by inhibiting crystallization from the supersaturated solution generated by dissolution of the amorphous material. Furthermore, understanding the mechanism of dissolution rate enhancement is of vital importance. In this work, wetting kinetics was taken up as an alternative approach for understanding the enhanced dissolution rate for amorphous solid dispersion of a poorly soluble drug. While cilostazol (CIL) was selected as the model drug, povidone (PVP), copovidone, and hypromellose (HPMC) were the polymers of choice. The concentrations against time profiles were evaluated for the supersaturated solutions of CIL in the presence and absence of the selected polymers. The degree of supersaturation increased significantly with increase in polymer content within the solid dispersion. While povidone was found to maintain the highest level of supersaturation for the greatest length of time both in dissolution and solution crystallization experiments, copovidone and hypromellose were found to be the less effective as crystallization inhibitor. The ability of polymers to generate and maintain supersaturated drug solutions was assessed by dissolution studies. The wetting kinetics was compared against the solid dispersion composition to establish a correlation with enhanced dissolution rate.KEY WORDS: Cilostazol, Crystallization inhibition, Solid dispersions, Supersaturated solutions, Wetting kinetics     

Development and Characterisation of Ursolic Acid Nanocrystals Without Stabiliser Having Improved Dissolution Rate and In Vitro Anticancer Activity

Ursolic acid (UA), which is a natural pentacyclic triterpenoid, has the potential to be developed as an anticancer drug, whereas its poor aqueous solubility and dissolution rate limit its clinical application. The aim of the present study was to develop UA nanocrystals to enhance its aqueous dispersibility, dissolution rate and anticancer activity. Following the investigation on the effects of stabiliser, the ratio of organic phase to aqueous solution and drug concentration, the UA nanocrystals without stabiliser were successfully prepared by anti-solvent precipitation approach. The nanocrystals maintained similar crystallinity with particle size, polydispersion index and zeta potential values of 188.0 ± 4.4 nm, 0.154 ± 0.022, and −25.0 ± 5.9 mV, respectively. Compared with the raw material, the UA nanocrystals showed good aqueous dispensability and a higher dissolution rate, and they could be completely dissolved in 0.5% SDS solution within 120 min. Moreover, the suspension of UA nanocrystals was physically stable after storage at 4°C for 7 weeks. By inducing G₂/M phase cell cycle arrest, the UA nanocrystals significantly induced stronger cell growth inhibition activity against MCF-7 cells compared with free drug in vitro, although the uptake of free UA was approximately twice higher than that of the UA nanocrystals. The UA nanocrystals may be used as a potential delivery formulation for intravenous injection with enhanced dissolution velocity and anticancer activity.Key words: anticancer, dissolution, MCF-7, nanocrystals, ursolic acid     

On-line detection of atmospheric formaldehyde by a conductometric biosensor

Atmospheric formaldehyde (CH(2)O) was detected under continuous flow conditions by an on-line system comprising of a wet scrubber for a continuous transfer of the pollutant to an aqueous solution, a micro-reactor containing immobilized formaldehyde dehydrogenase (FDH) and a conductometric transducer. By this system atmospheric formaldehyde concentrations in the range 0.05-2 ppm were detected with a sensitivity of 20 microS/ppm. In this concentration range the immobilized enzyme oxidized all the sampled formaldehyde molecules to formic acid, avoiding cumbersome calibration procedures. The operational stability of the biosensor was at least 3 months, working continuously 10 h/day at room temperature.     

Wet spinning of silk polymer. II. Effect of drawing on the structural characteristics and properties of filament

Regenerated silk fibroin (SF) filaments were prepared by the wet spinning technique. The rheological behavior of the SF dope solution prepared with formic acid was examined and the drawing effect on the structural characteristics and mechanical properties of SF filament was comparatively studied with those of natural silk fiber. SF dope exhibited shear thinning, but, as the dope concentration increased, the effect of shear thinning decreased, an indication that a higher concentration of dope solution will result in good spinnability. Wet-spun SF filaments exhibited a uniform and circular cross-sectional shape and dense morphology under SEM observation. X-ray diffraction (XRD) results revealed that the crystallinity of wet-spun regenerated filaments was hardly affected by the draw ratio, whereas the crystalline and amorphous orientation of regenerated SF filament showed different features depending on the drawing. The crystalline orientation of regenerated filaments increased with an increase of draw ratio and was lower than that of natural silk fiber. On the contrary, the amorphous orientation was constant throughout 1X-5X draw ratios, after an abrupt increase at 1X, and was higher than that of natural silk fiber. These differences in the orientation behaviors are attributed to the different spinning mechanisms involved. The tensile property was strongly dependent on the draw ratio. The breaking strength and elongation of the regenerated filament at 5X draw ratio were 2.2 g/day and 17%, respectively.     

Propagation of Biochirality: Crossovers and Nonclassical Crystallization Kinetics of Aspartic Acid in Water

All experimental procedures discussed could be treated as a screening tool for probing the existence of molecular association among the chiral molecules and the solvent system. The molecular association phases of a racemic conglomerate solution (CS) and a racemic compound solution (RCS), and the templating effect of aspartic acid solid surface were observed to minimize the chance of redissolving racemic conglomerate and racemic compound aspartic acid in water and reforming an RCS in crossovers experiments. Only 1 %wt% of l‐aspartic acid was adequate enough to induce a transformation from a racemic compound aspartic acid to a racemic conglomerate aspartic acid. This would make the propagation of biochirality more feasible and sound. However, tetrapeptide, (l‐aspartic acid)₄, failed to induce enantioseparation as templates purely by crystallization. Nonclassical crystallization theory was needed to take into account the existence of a CS. Fundamental parameters of the crystallization kinetics such as the induction time, interfacial energy, Gibbs energetic barrier, nucleation rate, and critical size of stable nuclei of: (i) racemic compound aspartic acid, (ii) racemic compound aspartic acid seeded with 1 %wt% l‐aspartic acid, (iii) racemic conglomerate aspartic acid, and (iv) l‐aspartic acid were evaluated and compared with different initial supersaturation ratios. Morphological studies of crystals grown from the crystallization kinetics were also carried out.Chirality 25:768–779, 2013. © 2013 Wiley Periodicals, Inc.     

Concerning the pathway from 19-oxoandrost-4-ene-3,17-dione to estrone

The conversion of a molecule of 19-oxoandrost-4-ene-3,17-dione [1a] to estrone [2a] by human placental aromatase requires a molecule of oxygen and of NADPH. An atom of this molecule of oxygen is incorporated into the extruded formic acid derived from C-19 of [1a]. It was proposed that the 0₂ is utilized for the enzymatic 2β-hydroxylation of [1a] and the released intermediate 2β-hydroxy-19-oxoandrost-4-ene-3, 17-dione [5a]aromatizes nonenzymatically. Should [5a] be an obligatory intermediate of estrogen biosynthesis, then all the oxygen of its 2β-hydroxyl must be incorporated into the extruded formic acid. We have previously synthesized [2β-¹⁸0;19-³H][5c] and proved that none of its 2β-¹⁸0 was incorporated in the formic acid extruded in the aromatization. On this basis we concluded that [5a] can not be an obligatory precursor of estrogen biosynthesis.

The trapping of radioactive androst-4-ene-2β,3β,17β,19-tetrol in a reductively terminated incubation of a mixture of radioactive androst-4-ene-3, 17-dione and [5a] with crude placental aromatase was interpreted as evidence in support of the intermediacy of [5a]. We confirmed that the tetrol can indeed be trapped in the reductively terminated incubations. However, considering that the crude placental enzyme preparation very likely contains numerous activated oxygen species capable of a variety of oxidation reactions, most of which may not be related to estrogen elaboration, and in view of our results quoted above, the origin and the eventual biosynthetic role of the parent compound of the tetrol remains to be determined.     

Variability in ionization state,stoichiometry and aggregation in histidine complexes with formic acid

The crystal structures of the complexes of L and DL histidine with formic acid have been determined as part of an effort to define biologically and evolutionarily important interactions and aggregation patterns. In terms of ionization state and stoichiometry they may be described as L-histidine formate formic acid and DL-histidine formate monohydrate respectively. In the L-histidine complex, amino acid molecules arranged in head-to-tail sequences centred around 2₁ screw axes are interconnected by formic acid molecules and formate ions. Histidine-formate interactions in the structure gives rise to a characteristic interaction pattern involving a linear array of alternating imidazole groups and formate ions. In DL-histidine formale monohydrate, head-to-tail sequences involving glide related molecules are interconnected through main chain-side chain interactions leading to amino acid layers. The layers are held together by formate ions and water molecules arranged in strings along which the ion and the molecule alternate. The patterns of amino acid aggregation in histidine complexes exhibit considerably higher variability than those in complexes involving arginine and lysine do. X-ray studies on crystalline complexes involving amino and peptides Part XXIX.