Aqueous Silk Fibroin Solution Research Articles

Preparation of Small-Diameter Silk Fibroin Tubular Scaffolds with Electrospinning Method

In recent years, electrospinning has received much attention to prepare porous tubular scaffold due to electrospun nanofibers mat, which can mimic the structure characteristics of the extracellular matrix (ECM). In the present paper, silk fibroin (SF) tubular scaffolds with small-diameter were prepared by electrospinning from regenerated silk fibroin (RSF) aqueous solutions with rotating mandrel collector. The morphology and fiber diameter distribution of SF tubular scaffolds were influenced much by the concentration of regenerated silk fibroin (RSF) solution, collecting distance and mandrel diameter. The results showed that SF tubular scaffold, which was in flat surface with smaller fiber diameter and uniform distributed, could be obtained at the concentration of 22%, collection distance of 10 cm and mandrel diameter of 4 mm. The structure of SF tubular scaffolds before and after ethanol treatment was characterized by FTIR and XRD. After ethanol treatment, the SF tubular scaffolds had a conformation transition from random-coil conformation to Silk II conformation.

Coimmobilization of Naringinases on Silk Fibroin Nanoparticles and Its Application in Food Packaging

Bombyx mori silk fibroin is a macromolecular biopolymer with remarkable biocompatibility. It was degummed and subjected to a series of treatments, including dissolution and dialysis, to yield an aqueous solution of silk fibroin, which was introduced rapidly into excess acetone to produce crystalline silk fibroin nanoparticles (SFNs), which were conjugated covalently with naringinase using glutaraldehyde as the cross-linking reagent. The SFN naringinases are easily recovered by centrifugation and can be used repeatedly. Naringinase is a bienzyme consisting of α-L-rhamnosidase and flavonoid-β-glucosidase. The enzyme activity and its kinetics were similar to those of the native form, and the optimum reactive temperature for both is 55°C. In our study, centrifugation allowed the separation of enzyme and substrate; after eight cycles the SFN naringinases retained >70% residual activity. The highly efficient processing technology and the use of SFN as a novel vector for a bienzyme have great potential for research and the development of food processing such as the debittering of naringin-containing juices.

Influence of the protocol used for fibroin extraction on the mechanical properties and fiber sizes of electrospun silk mats

Silk fibroin (SF) was regenerated using three of the most common protocols described in the bibliography for the dissolution of raw SF (LiBr 9.3M, CaCl2 50wt.% or CaCl2:EtOH:H2O 1:2:8 in molar ratio). The integrity of regenerated SF in aqueous solution was analyzed by SDS-PAGE and different profiles of degradation were observed depending on the protocol used. This fact was found to affect also the aqueous solubility of the freeze dried protein. These different SFs were used to produce electrospun mats using SF solutions of SF 17wt.% in 1,1,1,1′,1′,1′-hexafluoro-2-propanol (HFIP) and significant differences in fiber sizes, elongation and ultimate strength values were found. This work provides a global overview of the manner that different methods of SF extraction can affect the properties of electrospun SF-mats and consequently it should be considered depending on the use they are going to be made for.

Ultrasonication-Induced Rapid Gelation of Wild Silkworm Silk Fibroins

Silk fibroin （SF） hydrogels of the wild silkworm species Antheraea pernyi and Antheraea yamamai were obtained from aqueous SF solutions at room temperature. Both A. pernyi and A. yamamai solutions were slow to gelate. Hydrogels of the two species of wild silkworm were obtained rapidly following ultrasonicaton at 400–500 W. The secondary structure of the freeze-dried SF hydrogels was measured by X-ray diffraction and Fourier transform infrared spectroscopy. Ultrasonication did not change the main secondary structure of the hydrogels, but it accelerated the structural transformation of silk fibroin molecules from random coil or α helix to β sheet and reduced the gelation time.

A belt-like superfine film fabricated by the bubble-electrospinning

A belt-like superfine film is prepared via the bubble-electrospinning from an aqueous silk fibroin solution. The morphology of film structures is characterized by the scanning electron microscope. The average width of the film is about one micrometer and the thickness is about 377 nanometers. <br><br><font color='red'><b>This article has been retracted. Link to the retraction <u><a href='http://dx.doi.org/10.2298/TSCI150120012E'>10.2298/TSCI150120012E</a><u></b></font>

Gelation of <i>Antheraea pernyi</i> Silk Fibroin Accelerated by Shearing

The rapid manufacture of silk fibroin gels in mild conditions is an important subject in the field of silk-based biomaterials. In this study, the gelation of Antheraea pernyi silk fibroin (ASF) aqueous solution was induced by shearing, without chemical cross-linking agents. Simple shearing controlled and accomplished the steady and rapid conformational transition to β-sheets with ease. The conformational transformation and rapid gelation mechanisms of ASF induced by shearing were tracked and analyzed by circular dichroism spectrometry, Fourier transform infrared spectroscopy and X-ray diffractometry, then compared with Bombyx mori silk fibroin (BSF). ASF quickly formed hydrogels within 24 - 48 h after shearing under different shearing rates for 30 - 90 min, resulting in sol-gel transformation when the β-sheet content reached nearly 50%, which is the minimum content needed to maintain a stable hydrogel system in ASF. The gel structures remained stable once formed. The rapid gelation of ASF through shearing compared with BSF was achieved because of ASF’s alternating polyalanine-containing units, which tend to form α-helix structures spontaneously. Further, the entropic cost during the conformational transition from the α-helix to the β-sheet structure is less than the cost of the transition from the random coil structure. This method is a simple, non-chemical cross-linking approach for the promotion of rapid gelation and the protection of the biological properties of ASF, and it may prove useful for application in the field of biomedical materials.

Synthesis of hierarchical three-dimensional copper oxide nanostructures through a biomineralization-inspired approach

Three-dimensional (3D) copper oxide (CuO) nanostructures were synthesized in a regenerated Bombyx mori silk fibroin aqueous solution at room temperature. In the synthesis process, silk fibroin served as the template and helped to form the hierarchical CuO nanostructures by self-assembly. Cu(OH)2 nanowires were formed initially, and then they transformed into almond-like CuO nanostructures with branched edges and a compact middle. The size of the final CuO nanostructures can be tuned by varying the concentration of silk fibroin in the reaction system. A possible mechanism has been proposed based on various characterization techniques, such as scanning and transmission electron microscopy, X-ray diffraction, and thermogravimetric analysis. The synthesized CuO nanostructured material has been evaluated as an anode material for lithium ion batteries, and the result showed that they had a good electrochemical performance. The straightforward energy-saving method developed in this research may provide a useful preparation strategy for other functional inorganic materials through an environmentally friendly process.

Preparation of regenerated silk fibroin/silk sericin fibers by coaxial electrospinning

The coaxial electrospinning using the regenerated silk fibroin (SF) and silk sericin (SS) aqueous solutions as the core and shell spinning dopes, respectively, was carried out to prepare regenerated SF/SS composite fibers with components and core–shell structure similar to the natural silkworm silks. It was found from the scanning electron microscope (SEM) and transmission electron microscope (TEM) results that the core dope (SF aqueous solution) flow rate (Qc) and the applied voltage (V) had some effects on the morphology of the composite fiber. With an increase in Qc, the diameter nonuniformity and eccentricity of the core fiber became serious, while the increasing V played an inverse role. In this work, the suitable Qc for the fiber formation with better electrospinnability was about 6μL/min, and the corresponding optimum V was 40kV. Moreover, the results from Raman spectra analysis, modulated differential scanning calorimetry (MDSC), thermogravimetry (TG) measurement and mechanical property test showed that, compared with the pure SF fiber, the coaxially electrospun SF/SS fiber had more β-sheet conformation, better thermostability and mechanical properties. This was probably because that SS played significant roles in dehydrating SF molecules and inducing the conformational transition of SF to β-sheet structure.

Preparation and characterization of electrospun Antheraea pernyi silk fibroin nanofibers from aqueous solution

AbstractAntheraea pernyi silk fibroin nanofibers were prepared from aqueous solution with electrospinning. The viscosity and conductivity values of the A. pernyi silk fibroin aqueous solutions with different concentrations were measured, and the morphologies of the prepared electrospun nanofibers were observed by scanning electron microscopy. The morphologies of the electrospun nanofibers changed from beadlike to uniform cylindrical and then to beltlike. The A. pernyi silk fibroin aqueous solution with a concentration of 31% exhibited good spinnability, in which uniform and regular nanofibers with an average diameter of 422 nm were obtained. The secondary structure of the electrospun A. pernyi silk fibroin nanofibers was studied by solid 13C‐NMR cross‐polarization/magic angle spinning spectroscopy, X‐ray diffraction, and differential scanning calorimetry. The as‐spun A. pernyi silk fibroin nanofibers presented a complete α‐helix/random coil structure, and no β‐sheet structure was observed. After insoluble treatment with water vapor, α‐helix and β‐sheet conformations coexisted in the A. pernyi silk fibroin nanofibers, and the β‐sheet conformation was predominant. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

A Natural Silk Fibroin Protein‐Based Transparent Bio‐Memristor

AbstractThe recent discovery of nanoelectronics memristor devices has opened up a new wave of enthusiasm and optimism in revolutionizing electronic circuit design, marking the beginning of new era for the advancement of neuromorphic, high‐density logic and memory applications. Here a highly non‐linear dynamic response of a bio‐memristor is demonstrated using natural silk cocoon fibroin protein of silkworm, Bombyx mori. A film that is transparent across most of the visible spectrum is obtained with the electronic‐grade silk fibroin aqueous solution of ca. 2% (wt/v). Bipolar memristive switching is demonstrated; the switching mechanism is confirmed to be the filamentary switching as observed by probing local conduction behavior at nanoscale using scanning tunneling microscopy. The memristive transition is elucidated by a physical model based on the carrier trapping or detrapping in silk fibroin films and this appears to be due to oxidation and reduction procedures, as evidenced from cyclic voltammetry measurements. Hence, silk fibroin protein could be used as a biomaterial for bio‐memristor devices for applications in advanced bio‐inspired very large scale integration circuit design as well as in biologically inspired synapse links for energy‐efficient neuromorphic computing.

Preparation of Porous Scaffolds from Silk Fibroin Extracted from the Silk Gland of Bombyx mori (B. mori)

In order to use a simple and ecofriendly method to prepare porous silk scaffolds, aqueous silk fibroin solution (ASF) was extracted from silk gland of 7-day-old fifth instar larvae of Bombyx mori (B. mori). SDS-page analysis indicated that the obtained fibroin had a molecular weight higher than 200 kDa. The fabrication of porous scaffolds from ASF was achieved by using the freeze-drying method. The pore of porous scaffolds is homogenous and tends to become smaller with an increase in the concentration of ASF. Conversely, the porosity is decreased. The porous scaffolds show impressive compressive strength which can be as high as 6.9 ± 0.4 MPa. Furthermore, ASF has high cell adhesion and growth activity. It also exhibits high ALP activity. This implies that porous scaffolds prepared from ASF have biocompatibility. Therefore, the porous scaffolds prepared in this study have potential application in tissue engineering due to the impressive compressive strength and biocompatibility.

Ultrasonication and Genipin Cross-Linking to Prepare Novel Silk Fibroin–Gelatin Composite Hydrogel

Hydrogels are highly desirable tissue engineering scaffolds due to their high water content and structural similarity to a natural extra cellular matrix. However, the extensive use of hydrogels is limited by their low strength and facile degradation. By combining mechanical integrity and slow degradation of silk fibroin with excellent bioactivity of gelatin, a novel biocompatible protein-based composite hydrogel of silk fibroin and gelatin was developed. The gelation of silk fibroin aqueous solution was accelerated by ultrasonication, and gelatin derived from porcine skin was immobilized into the hydrogel network by the silk fibroin β-sheets. After that, genipin was used to post-cross-link the hydrogel to form a compact and stable hydrogel network. This hydrogel composite was a mechanically robust biomaterial with predictable long-term degradation characteristics. MG63 cells readily attached, spread, and proliferated on the surface of the hydrogels as demonstrated by fluorescein diacetate/propidium iodide staining and mitochondrial activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Furthermore, the physicochemical and biological properties of hydrogel were fine-tunable by altering the ratio of silk fibroin and gelatin. The silk fibroin/gelatin composite hydrogels are anticipated to have the potential as cartilage or non-load-bearing bone tissue engineering and regeneration.

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 CaCl2/H2O/EtOH solution for dissolution times ranging from 3 to 180min, 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 10–30nm particle size, whereas the three SFC samples exhibited a turbid solution with 100–300nm 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 (0.1–3s−1).

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.

抗菌丝素蛋白水凝胶敷料的制备<br>Preparation of Antimicrobial Silk Hydrogels for Surgical Dressings

季铵盐类抗菌剂作为凝胶促进剂，诱导丝素蛋白水溶液快速原位凝胶化从而制备抗菌性丝素水凝胶敷料。利用X射线衍射、红外光谱、扫描电镜、zeta电位分析、差热分析等手段对所制丝素水凝胶的结构、zeta电位、热稳定、降解性及其抗菌性能进行了表征。研究结果表明，季铵盐会引起丝素颗粒负电位值下降，由此促发形成的丝素水凝胶为β-折叠片层结构，凝胶内部形貌为多孔的三维网状结构，且对革兰氏阳性菌和阴性菌都有很明显的抗菌效果。同时由体外降解、差热分析结果可知，季铵盐引发丝素蛋白溶液快速形成水凝胶，蛋白酶可以在体外降解水凝胶。季铵盐在水凝胶中主要以无定形或亚稳态晶体状态存在，容易扩散释放，达到较好的抗菌效果。抗菌性原位丝素蛋白水凝胶预计可以用于外科伤口敷料。 Using quaternary ammonium salts (QAS) as initiating agents, silk fibroin aqueous solutions were induced to gel rapidly. The structure, zeta-potential change and thermal stability properties of QAS-induced silk gel were charac- terized by means of X-ray diffraction (XRD), infrared spectroscopy (IR), scanning electron microscopy (SEM), zeta- potential measurements and differential thermal analysis (DTA). The results show that QAS-induced silk gelation is accompanied by the formation of β-sheets and less negative zeta-potential values. With the observation of SEM, the internal morphology of gel exhibited a porous three-dimensional network structure. In vitro degradation and DTA analysis revealed that QAS-induced fibroin gel was not quite stable and disintegrated more easily in the enzyme solu- tion. Also, QAS is amorphous or metastable crystal state in silk gel and thus easy to release and diffuse. The antibacte- rial test in vitro suggested that silk-QAS gel showed distinctly antibacterial activities against both Gram-positive and Gram-negative bacteria. In situ antimicrobial silk hydrogel is expected to be used for a surgical dressing.

抗菌丝素蛋白水凝胶敷料的制备

季铵盐类抗菌剂作为凝胶促进剂，诱导丝素蛋白水溶液快速原位凝胶化从而制备抗菌性丝素水凝胶敷料。利用X射线衍射、红外光谱、扫描电镜、zeta电位分析、差热分析等手段对所制丝素水凝胶的结构、zeta电位、热稳定、降解性及其抗菌性能进行了表征。研究结果表明，季铵盐会引起丝素颗粒负电位值下降，由此促发形成的丝素水凝胶为β-折叠片层结构，凝胶内部形貌为多孔的三维网状结构，且对革兰氏阳性菌和阴性菌都有很明显的抗菌效果。同时由体外降解、差热分析结果可知，季铵盐引发丝素蛋白溶液快速形成水凝胶，蛋白酶可以在体外降解水凝胶。季铵盐在水凝胶中主要以无定形或亚稳态晶体状态存在，容易扩散释放，达到较好的抗菌效果。抗菌性原位丝素蛋白水凝胶预计可以用于外科伤口敷料。 Using quaternary ammonium salts (QAS) as initiating agents, silk fibroin aqueous solutions were induced to gel rapidly. The structure, zeta-potential change and thermal stability properties of QAS-induced silk gel were charac- terized by means of X-ray diffraction (XRD), infrared spectroscopy (IR), scanning electron microscopy (SEM), zeta- potential measurements and differential thermal analysis (DTA). The results show that QAS-induced silk gelation is accompanied by the formation of β-sheets and less negative zeta-potential values. With the observation of SEM, the internal morphology of gel exhibited a porous three-dimensional network structure. In vitro degradation and DTA analysis revealed that QAS-induced fibroin gel was not quite stable and disintegrated more easily in the enzyme solu- tion. Also, QAS is amorphous or metastable crystal state in silk gel and thus easy to release and diffuse. The antibacte- rial test in vitro suggested that silk-QAS gel showed distinctly antibacterial activities against both Gram-positive and Gram-negative bacteria. In situ antimicrobial silk hydrogel is expected to be used for a surgical dressing.

Comparison of Gelation Time and Polyalcohol Effect on Hydrogels from Domestic and Wild Silk Fibroins

Silk fibroin (SF) hydrogels were obtained from both domestic (Bombyx mori) and wild (Antheraea pernyi) silkworms from aqueous silk fibroin solutions at room temperature. The gelation time of theAntheraea pernyi(A. pernyi) SF solution was significantly shorter than that of theBombyx mori(B. mori) SF solution. The secondary structures of the two kinds of hydrogels were also compared. In order to further reduce the gelation time, various amounts of polyethylene glycol (PEG) were blended with the silk fibroins ofA. pernyiandB. mori. The gelation time of bothA. pernyiSF andB. moriSF decreased with the increased amount of PEG. After freeze-drying, the hydrogels were characterized through X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and Raman spectroscopy. Results showed that the addition of polyalcohol did not change the main secondary structure of the hydrogels. However, the addition of polyalcohol did reduce the gelation time and triggered additional formation ofβ-sheets.

Nanoparticles fabricated by the bubble electrospinning

The bubble electrospinning is used to produce nanoparticles from several nano-meters to several hundred nanometers in diameter. Silk fibroin aqueous solution with low concentration is used in the experiment. The spinning process can be well controlled by temperature.

Ion electrodiffusion governs silk electrogelation

Silk electrogelation involves the transition of an aqueous silk fibroin solution to a gel state (E-gel) in the presence of an electric current. The process is based on local pH changes as a result of water electrolysis - generating H(+) and OH(-) ions at the (+) and (-) electrodes, respectively. Silk fibroin has a pI=4.2 and when local pH<pI, E-gel forms. An experimental system was constructed that allowed the measurement of E-gel growth and pH distribution for an applied current. To explain the observed rectangular pH profile of pH(gel)~4 surrounded by pH(silk-solution)~10, a finite-element ion electrodiffusion model was developed. The model relies on electrodiffusion of the generated H(+) and OH(-) ions. Initially, inputs into the model were the measured E-gel and voltage curves. The governing ion electrodiffusion equations were solved and the calculated pH matched the experimental pH profile, indicating that ion electrodiffusion dictates local pH changes and E-gel growth. Furthermore, the model predicted the constant currents (2mA and 3mA) necessary for two hypothetical E-gel growth curves and these results were then validated experimentally. The model thus shows how ion electrodiffusion governs the electrogelation process and also provides predictable outcomes for fundamental and practical E-gel applications.

A bio-inspired microfluidic concentrator for regenerated silk fibroin solution

In this article, microfluidic channels (500μm in width, 65–85μm in depth) in micro-chips were fabricated in poly-(dimethylsiloxane) (PDMS) on a mold of SU-8 photoresist based on photolithography process. By biomimicking the silk glands and the spinning duct of silkworms, the chips were designed and used for the concentration of regenerated silk fibroin (RSF) aqueous solution. The microfluidic devices based on regenerated cellulose membrane involve a flow of RSF aqueous solution on a donor side and a flow of polyethyleneglycol (PEG) aqueous solution on an acceptor side. The enrichment factor was characterized by varying the flow rates of the RSF solution and the PEG solution, and the concentrations of PEG and RSF solutions at inlets. The results showed that the enrichment efficiency varied as the flow factors changed. The RSF concentration of the solution was enriched up to 31.2wt% from 12wt% by using the microfluidic chip. The experiments facilitated the enrichment of RSF solution in a microfluidic channel as silkworm did. It might be further applied for the microfluidic spinning of RSF fiber.