Effect of degumming time on silkworm silk fibre for biodegradable polymer composites

Recently, many studies have been conducted on exploitation of natural materials for modern product development and bioengineering applications. Apart from plant-based materials (such as sisal, hemp, jute, bamboo and palm fibre), animal-based fibre is a kind of sustainable natural materials for making novel composites. Silkworm silk fibre extracted from cocoon has been well recognized as a promising material for bio-medical engineering applications because of its superior mechanical and bioresorbable properties. However, when producing silk fibre reinforced biodegradable/bioresorbable polymer composites, hydrophilic sericin has been found to cause poor interfacial bonding with most polymers and thus, it results in affecting the resultant properties of the composites. Besides, sericin layers on fibroin surface may also cause an adverse effect towards biocompatibility and hypersensitivity to silk for implant applications. Therefore, a proper pre-treatment should be done for sericin removal. Degumming is a surface modification process which allows a wide control of the silk fibre's properties, making the silk fibre possible to be used for the development and production of novel bio-composites with unique/specific mechanical and biodegradable properties. In this paper, a cleaner and environmentally friendly surface modification technique for tussah silk in polymer based composites is proposed. The effectiveness of different degumming parameters including degumming time and temperature on tussah silk is discussed through the analyses of their mechanical and morphological properties. Based on results obtained, it was found that the mechanical properties of tussah silk are affected by the degumming time due to the change of the fibre structure and fibroin alignment.     

Preparation and mechanical properties of layers made of recombinant spider silk proteins and silk from silk worm

Layers of recombinant spider silks and native silks from silk worms were prepared by spin-coating and casting of various solutions. FT-IR spectra were recorded to investigate the influence of the different mechanical stress occurring during the preparation of the silk layers. The solubility of the recombinant spider silk proteins SO1-ELP, C16, AQ24NR3, and of the silk fibroin from Bombyx mori were investigated in hexafluorisopropanol, ionic liquids and concentrated salt solutions. The morphology and thickness of the layers were determined by Atomic Force Microscopy (AFM) or with a profilometer. The mechanical behaviour was investigated by acoustic impedance analysis by using a quartz crystal microbalance (QCMB) as well as by microindentation. The density of silk layers (d<300 nm) was determined based on AFM and QCMB measurements. At silk layers thicker than 300 nm significant changes of the half-band-half width can be correlated with increasing energy dissipation. Microhardness measurements demonstrate that recombinant spider silk and sericine-free Bombyx mori silk layers achieve higher elastic penetration modules E_EP and Martens hardness values H_M than those of polyethylenterephthalate (PET) and polyetherimide (PEI) foils.     

Silver nanoparticle containing silk fibroin bionanotextiles

Development of new generation bionanotextiles is an important growing field, and they have found applications as wound dressings, bandages, tissue scaffolds, etc. In this study, silver nanoparticle (AgNP) containing silk-based bionanotextiles were fabricated by electrospinning, and processing parameters were optimized and discussed in detail. AgNPs were in situ synthesized within fibroin nanofibers by UV reduction of silver ions to metallic silver. The influence of post-treatments via methanol treatment and glutaraldehyde (GA) vapor exhibited changes in the secondary structure of silk. Methanol treatment increased the tensile properties of fibers due to supported crystalline silk structure, while GA vapor promoted amorphous secondary structure. AgNP containing silk fibroin bionanotextiles had strong antibacterial activity against gram-positive Staphylococcus aureus and gram-negative Pseudomonas aeruginosa.     

Silk peptide production from whole silkworm cocoon using ultrasound and enzymatic treatment and its suppression of solar ultraviolet-induced skin inflammation

Silk fibroin, which is derived from sericin through degumming, is mainly used as a biomaterial. However, interest in functional verification and industrial applications of sericin has been growing for several years. We used ultrasonication to simplify the extraction process of the silk peptide under low salt conditions at 20 °C, instead of using the conventional conditions of high salt and temperature. The concentration of the silk peptide was measured to determine the optimized extraction time and solvent, which were 4 h and 0.1 N NaOH, respectively. The molecular weight of the enzyme-treated silk peptide was measured using SDS-PAGE and GPC. Silk peptide treated with papain after ultrasound had a molecular weight of less than 5 kDa, and the papain treated-silk peptide reduced solar ultraviolet-induced COX-2 expression through inhibition of ERK phosphorylation. This is the first study investigating simultaneous extraction of fibroin and sericin, which can be used for mass production of food materials.     

Microfluidic Fabrication of Multi‐Drug‐Loaded Polymeric Microparticles for Topical Glaucoma Therapy

In this paper, the fabrication and characterization of multi‐drug‐loaded microparticles are demonstrated for topical glaucoma therapy. Specifically, latanoprost (“LAT”) and dexamethasone (“DEX”) are loaded in monodisperse microparticles (diameter ≈150 μm) of a biodegradable polymer–poly (lactic‐co‐glycolic) acid (PLGA)—using capillary microfluidics coupled with solvent evaporation. Both individual (LAT in PLGA and DEX in PLGA) and combined (LAT and DEX in PLGA) microparticle formulations are demonstrated. The morphology, size distribution and in vitro release kinetics are studied, and in vitro mucoadhesion of the formulated microparticles is also assessed. In addition, discussion is placed in how precise knowledge of the particle composition enabled by the microfluidic fabrication method and in vitro release rate measurements allow for facile topical formulation design and dose optimization. Such precision‐fabricated, multi‐drug loaded, sustained‐release microparticles are envisioned to serve as a promising platform for topical administration of ocular drugs. This could potentially reduce the frequency of eyedrop‐based drug administration from several times a day to merely once a day (or less), thus greatly facilitating patient compliance and adherence to a strict therapeutic drug regimen.     

Raman and surface enhanced Raman scattering of a black dyed silk

The Raman and surface enhanced Raman scattering (SERS) spectra of a black dyed silk sample (BDS) were registered. The spectral analysis was performed on the basis of Raman and SERS spectral data of isolated samples of Bombyx mori silk fibroin, its motif peptide component (GAGAGS) and the synthetic reactive black 5 dye (RB5). The macro FT‐Raman spectrum of the silk sample is consistent with a silk II‐Cp crystalline fraction of Bombyx mori silk fibroin; the SERS spectrum is highly consistent with conformational modifications of the fibroin due to the interactions with the Ag nanoparticles. The GAGAGS peptide sequence dominates the Raman spectrum of the silk. The SERS spectrum of the peptide suggests a random coil conformation imposed by the surface interaction; the serine residue in the new conformation is exposed to the surface. Quantum chemical calculations for a model of the GAGAGS–Ag surface predict a nearly extended conformation at the Ag surface. The Raman spectrum of the dye was analysed, and a complete band assignment was proposed; it was not possible to propose a preferential orientation or organization of the molecule on the metal surface. Quantum chemical calculations for a model of the dye interacting with a silver surface predict a rather coplanar orientation of the RB5 on the Ag metal surface. The Raman spectrum of the BDS sample is dominated by signals from the dye; the general spectral behaviour indicates that the dye mainly interacts with the silk through the sulphone (–SO₂–) and sulphonate (–SO₂–O–) groups. Besides the presence of dye signals, mainly ascribed to the sulphone and sulphonate bands, the SERS spectrum of the BDS sample also displays bands belonging to the amino acids alanine, glycine, serine and particularly tyrosine. Copyright © 2013 John Wiley & Sons, Ltd.     

Processing and modification of films made from recombinant spider silk proteins

Protein films represent an interesting class of materials with various possibilities for applications. We investigated films made of two different synthetic spider silk proteins derived from the garden spider’s (Araneus diadematus) two dragline silk proteins ADF-3 and ADF-4. Protein films cast from hexafluoroisopropanol solutions displayed a predominantly α-helical secondary structure. Processing such films with potassium phosphate or methanol resulted in a transition to a β-sheet rich structure. While as-cast films could be dissolved in water, processed β-sheet rich films were water insoluble. The chemical stability of processed films depended on the amino acid sequence of the respective protein employed. As a proof of principle, fluorescent probes or enzymes were covalently attached to the film surface. The presented approach provides a basis for designing tailor-made protein films using silk proteins as scaffold, in which the film properties can be controlled by genetic engineering of the underlying silks. PACS 68.55.Jk; 81.15.Lm; 87.14.Ee     

Resilient silk captures prey in black widow cobwebs

The gumfoot thread of a black widow (Latrodectus hesperus) spider’s cob web is a spring-loaded trap that yanks walking insects into the web. Since spider silks are known as energy dissipating materials, we investigated this trap to find out where the energy is stored. Using previously measured material properties, we modeled the gumfoot thread as a damped harmonic oscillator and compared it to high speed video analysis of prey capture. These measurements show that the gumfoot thread is plastically deformed during prey capture and cannot be the site of energy storage. We then measured the material properties of scaffolding silk that makes up the upper portion of the cob web. Scaffolding silk is highly resilient (90%) at strains less than 3%. This energy storage is sufficient to drive the oscillations seen in prey capture and is consistent with the measured kinematics. This study is the first demonstration of energy-storage as a primary biological function for spider silk. PACS 87.15.La; 81.40.Jj; 81.05.Lg     

Formation of silk fibroin nanoparticles in water-miscible organic solvent and their characterization

When Silk fibre derived from Bombyx mori, a native biopolymer, was dissolved in highly concentrated neutral salts such as CaCl₂, the regenerated liquid silk, a gradually degraded peptide mixture of silk fibroin, could be obtained. The silk fibroin nanoparticles were prepared rapidly from the liquid silk by using water-miscible protonic and polar aprotonic organic solvents. The nanoparticles are insoluble but well dispersed and stable in aqueous solution and are globular particles with a range of 35–125 nm in diameter by means of TEM, SEM, AFM and laser sizer. Over one half of the ɛ-amino groups exist around the protein nanoparticles by using a trinitrobenzenesulfonic acid (TNBS) method. Raman spectra shows the tyrosine residues on the surface of the globules are more exposed than those on native silk fibers. The crystalline polymorph and conformation transition of the silk nanoparticles from random-coil and α-helix form (Silk I) into anti-parallel β-sheet form (Silk II) are investigated in detail by using infrared, fluorescence and Raman spectroscopy, DSC, ¹³C CP-MAS NMR and electron diffraction. X-ray diffraction of the silk nanoparticles shows that the nanoparticles crystallinity is about four fifths of the native fiber. Our results indicate that the degraded peptide chains of the regenerated silk is gathered homogeneously or heterogeneously to form a looser globular structure in aqueous solution. When introduced into excessive organic solvent, the looser globules of the liquid silk are rapidly dispersed and simultaneously dehydrated internally and externally, resulting in the further chain–chain contact, arrangement of those hydrophobic domains inside the globules and final formation of crystalline silk nanoparticles with β-sheet configuration. The morphology and size of the nanoparticles are relative to the kinds, properties and even molecular structures of organic solvents, and more significantly to the looser globular substructure of the degraded silk fibroin in aqueous solution. It is possible that the silk protein nanoparticles are potentially useful in biomaterials such as cosmetics, anti-UV skincare products, industrial materials and surface improving materials, especially in enzyme/drug delivery system as vehicle.     

Imaging contrast effects in alginate microbeads containing trapped emulsion droplets

This study focuses on spherical microparticles made of cross-linked alginate gel and microcapsules composed of an oil-in-water emulsion where the continuous aqueous phase is cross-linked into an alginate gel matrix. We have investigated the use of these easily manufactured microbeads as contrast agents for the study of the flow properties of fluids using nuclear magnetic resonance imaging. Results demonstrate that combined spin-spin (T(2)) relaxation and diffusion contrast in proton NMR imaging can be used to distinguish among rigid polymer particles, plain alginate beads, and alginate emulsion beads. Multi-echo CPMG spin-echo imaging indicates that the average spin-lattice (T(1)) and spin-spin (T(2)) relaxation times of the plain alginate and alginate emulsion beads are comparable. Meanwhile, diffusion-weighted imaging produces sharp contrast between the two types of alginate beads, due to restricted diffusion inside the embedded oil droplets of the alginate emulsion beads. While the signal obtained from most materials is severely attenuated under applied diffusion gradients, the alginate emulsion beads maintain signal strength. The alginate emulsion beads were added to a suspension and imaged in an abrupt, annular expansion flow. The emulsion beads could be clearly distinguished from the surrounding suspending fluid and rigid polystyrene particles, through either T(2) relaxation or diffusion contrast. Such a capability allows future use of the alginate emulsion beads as tracer particles and as one particle type among many in a multimodal suspension where detailed concentration profiles or particle size separation must be quantified during flow.     

Functionalized alginate/chitosan biocomposites consisted of cylindrical struts and biologically designed for chitosan release

A novel alginate/chitosan composite scaffold was developed. The composite scaffolds were fabricated at low temperature using a three-axis robot system connected to a micro-dispenser and a core/shell nozzle. The structure of the composite scaffolds included hollow struts; deposited chitosan on the inner walls (core region) of the struts reacted electrostatically with the alginate layer (shell region). The fabricated, highly porous composite scaffolds exhibited excellent mechanical properties and controllable chitosan release, which was closely dependent on the weight fraction of the alginate in the shell region. The tensile strength in the dry state was ∼1.8-fold greater than that of pure alginate scaffold due to the ionic interaction between alginate and chitosan. To determine the feasibility of using the developed scaffold in tissue regeneration applications, in vitro cellular responses were evaluated using osteoblast-like-cells (MG63). The cell proliferation on the composite scaffold was ∼3.4-fold greater than that on the pure alginate scaffold. Alkaline phosphate activity and calcium deposition of the composite scaffold after 14 and 21 days of cell culture were significantly enhanced (1.6- and 1.8-fold greater, respectively) compared with those of the pure alginate scaffold. These results suggested that the alginate/chitosan composite scaffolds with a controlled chitosan release have great potential for use in regenerating various tissues.     

Environmentally friendly surface modification of silk fiber: Chitosan grafting and dyeing

In this paper, the surface modification of silk fiber using anhydrides to graft the polysaccharide chitosan and dyeing ability of the grafted silk were studied. Silk fiber was degummed and acylated with two anhydrides, succinic anhydride (SA) and phthalic anhydride (PA), in different solvents (dimethyl sulfoxide (DMSO) and N,N-dimethyl formamide (DMF)). The effects of anhydrides, solvents, anhydride concentration, liquor ratio (L:R) and reaction time on acylation of silk were studied. The polysaccharide chitosan was grafted to the acylated silk fiber and dyed by acid dye (Acid Black NB.B). The effects of pH, chitosan concentration, and reaction time on chitosan grafting of acylated silk were investigated. The physical properties show sensible changes regardless of weight gain. Scanning electron microscopy (SEM) analysis showed the presence of foreign materials firmly attached to the surface of silk. FTIR spectroscopy provided evidence that chitosan was grafted onto the acylated silk through the formation of new covalent bonds. The dyeing of the chitosan grafted-acylated silk fiber indicated the higher dye ability in comparison to the acylated and degummed silk samples. The mechanism of chitosan grafting over degummed silk through anhydride linkage was proposed. The findings of this research support the potential production of new environmentally friendly textile fibers. It is worthwhile to mention that the grafted samples have antibacterial potential due to the antibacterial property of chitosan molecules.     

微乳液法制备海藻酸铁微粒

采用微乳液法制备了海藻酸铁微粒,并探索了表面活性剂用量、原料投量比、搅拌速度和时间等各种因素对海藻酸铁微粒粒径及形态的影响。研究结果表明,在表面活性剂质量与石油醚的体积比为1:5(g/mL)、海藻酸钠浓度2.0%、搅拌时间为3h以及搅拌速度为1000r/min的条件下,制得了平均粒径为9.25μm的海藻酸铁微粒。     

Comparative Raman spectroscopic analysis of orientation in fibers and regenerated films of Bombyx mori silk fibroin

This study is focused on the Raman spectroscopic analysis of degummed silk fibroin (SF) fibers and regenerated Bombyx mori silk fibroin films: a correlation was found between some spectral features related to the methylene deformation modes and the molecular orientation of the samples. Polarized Raman spectra on SF fibers were used to obtain the orientation distribution function of carbonyl groups along the protein backbone. The variation of the intensity ratio of 1400/1450 cm⁻¹ for the peaks attributed to the wagging and bending deformation modes of CH₂ groups with respect to the angular orientation of the fiber was measured and quantitatively correlated with the orientation distribution function of the carbonyl groups. Unpolarized Raman spectra were measured for regenerated silk fibroin films and lyophilized solutions. The variation of the intensity ratio of 1415/1455 cm⁻¹, which is related to the deformation modes of CH₂ groups in SF regenerated materials, was qualitatively related to the microstructural orientation of the samples observed by scanning electron microscopy (SEM), and to the presence of Silk I phase as suggested by the analysis of samples obtained in different casting conditions and also by the measurements on mechanically deformed films. The results obtained showed the utility of the spectroscopic intensity ratio of 1400/1450 cm⁻¹ for the rapid assessment of molecular orientation in silk fibers, which could be useful for quality and process control of regenerated silk‐based textiles. Moreover, the qualitative dependence of the intensity ratio of 1415/1455 cm⁻¹ was found to be sensitive to both the microstructural orientation and Silk I content of regenerated silk fibroin films, suggesting a possible correlation of this Raman marker of the Silk I phase with the degree of molecular order brought about by this polymorph. Copyright © 2006 John Wiley & Sons, Ltd.     

Supercritical fluid encapsulation of acizol into aliphatic polyether microparticles

Encapsulation of pharmaceutical grade acizol (Acizol® pharmaceutical substance) into bioresorbable D,L-polylactide and polylactoglycolide microparticles using supercritical carbon dioxide has been studied. An effective way for formation of polymer fine powders (mean particle size of about 10–20 µm) containing up to 20 wt % of the bioactive component without any organic solvent used has been suggested. Raman spectroscopy with spatial resolution was employed to analyze the distribution of acizol throughout the volume of the individual polymer microparticles and to study the kinetics of its release into saline. The rapid release (40–80% of the total amount of the encapsulated substance) from the samples under study was observed during the first hour, and then it was followed by a gradual, almost linear release between the 4th and 14th days of the experiment, with the total release continuing up to 100%.     

Tubuliform silk protein: A protein with unique molecular characteristics and mechanical properties in the spider silk fibroin family

Orb–web weavers can produce up to six different types of silk and a glue for various functions. Tubuliform silk is unique among them due to its distinct amino acid composition, specific time of production, and atypical mechanical properties. To study the protein composing this silk, tubuliform gland cDNA libraries were constructed from three orb–weaving spiders Argiope aurantia, Araneus gemmoides, and Nephila clavipes. Amino acid composition comparison between the predicted tubuliform silk protein sequence (TuSp1) and the corresponding gland protein confirms that TuSp1 is the major component in tubuliform gland in three spiders. Sequence analysis suggests that TuSp1 shares no significant similarity with its paralogues, while it has conserved sequence motifs with the most primitive spider, Euagrus chisoseus silk protein. The presence of large side-chain amino acids in TuSp1 sequence is consistent with the frustrated β-sheet crystalline structure of tubuliform silk observed in transmission electron microscopy. Repeat unit comparison within species as well as among three spiders exhibits high sequence conservation. Parsimony analysis based on carboxy terminal sequence shows that Argiope and Araneus are more closely related than either is to Nephila which is consistent with phylogenetic analysis based on morphological evidence. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. PACS 61.41; 87.14 Ee; 87.15 Cc     

Influence of grafting with acrylate compounds on the conformational rearrangements of silk fibroin upon electrospinning and treatment with aqueous methanol

Silk fabrics from Bombyx mori silkworm were grafted with 2‐hydroxyethyl methacrylate (HEMA) as well as a binary system of HEMA and 4‐hydroxybutyl acrylate (HBA) and then analysed by Raman and infrared (IR) spectroscopy to elucidate the interactions between the components and their possible conformational changes. The samples were then dissolved in trifluoroacetic acid and electrospun; the influence of the grafted polymers on the silk fibroin rearrangements upon these treatments was investigated by vibrational spectroscopy. Upon grafting, the fabrics underwent conformational rearrangements towards a more unordered state, although they kept their prevailing β‐sheet conformation; also the polymeric component underwent hydrogen bonding and backbone rearrangements upon interaction with silk fibroin and the occurrence of strong covalent bonds cannot be excluded. By immersing the as‐electrospun grafted and pure fibroin nanofibres (prevalently unordered) in aqueous methanol, they partially recovered the β‐sheet content observed in the corresponding starting fabrics; the percentage of recovery decreased along the series: pure silk > HEMA‐grafted silk > HEMA and HBA‐grafted silk. This trend suggests that the presence of the polyHEMA grafted component hinders the silk fibroin recrystallization into β‐sheet upon aqueous methanol treatment; moreover, the addition of the more sterically hindered HBA monomer in the grafting system further prevented this process. Copyright © 2016 John Wiley & Sons, Ltd.     

Solution behavior of synthetic silk peptides and modified recombinant silk proteins

Spider dragline silk from Nephila clavipes possesses impressive mechanical properties derived in part from repetitive primary sequence containing polyalanine regions that self-assemble into crystalline β-sheets. In the present study, we have sought to understand more details of redox responses related to conformational transitions of modified silk peptides and a recombinant protein containing encoded methionine triggers. Regardless of the position of the methionine trigger relative to the polyalanine domain, chemical oxidation was rapid and slight increases in the α-helical structure and decreases in the β-sheet and random coil content were observed by CD and FTIR in the assembled silk-like peptides and the recombinant protein. CD results indicated that the decrease in β-sheet and random coil conformations, coupled with the increase in helical content during oxidation, occurred during the first 30 min of the reaction. No further conformational changes occurred after this time and the response was independent of methionine trigger location relative to the penta-alanine domain. These results were confirmed with fluorescence studies. The design, processing and utility of these modified redox triggered silk-like peptides and proteins suggest a range of potential utility, from biomaterials to engineered surface coatings with chemically alterable secondary structure and, thus, properties. PACS 87.14.Ee; 87.64.-t; 87.6.+2.     

Structural changes of thin films from recombinant spider silk proteins upon post-treatment

Engineering of spider silk proteins offers the possibility to control their molecular sequence and thus their material properties. Spin coating was used to prepare films of engineered spider silk protein derived from the garden spider’s (Araneus diadematus) dragline silk protein ADF-4. A conformational transition from α-helix to β-sheet-rich structures upon methanol treatment of the films was detected by external reflection IR spectroscopy. We present direct evidence for this structural transformation using grazing-incidence X-ray diffraction (GIXRD) and small-angle scattering (GISAXS). The protein film structure after the methanol treatment consists mainly of β-sheet polyalanine crystals dispersed in an amorphous protein matrix. The GIXRD intensity profiles show Bragg peaks from β-sheet polyalanine crystallites having an average size of 7.5 nm. The non-uniform and large crystal size distributions within the film were explained based on the protein composition. The effect of the chemical nature of the interface on the protein film structure was investigated as well. PACS 87.15.-v; 68.55.-a; 61.10.Eq     

Patterning and photoluminescence of CdS nanocrystallites on silk fibroin fiber

CdS nanocrystallites could be formed and assembled into nanoparticle strings and hexagons on natural silk fibroin fiber (SFF) through a room-temperature bio-inspired process. Herein, the biomaterial SFF served as reactive substrate, not only provides the in situ formation sites for CdS nanocrystallites, but also directs the arrangement of nanocrystalline CdS simultaneously. The photoluminescence (PL) of the resulting nanocomposites CdS/SFF is investigated extensively. The PL peaks observed from CdS nanoparticle strings are similar to those of separate CdS nanoparticles, corresponding to the band-edge emission of their individual building blocks (QD-CdS). Moreover, CdS nanoparticle hexagons perform a red-shifted and broadened emission peak.