Fabrication and characterization of fibroin solution and nanoparticle from silk fibers of Bombyx mori

The present investigation aims to study the effect of degumming time on the structural property of silk fiber obtained by silk cocoons of Bombyx mori, followed by preparation of the regenerated silk fibroin (RSF) solution which can be subsequently molded into silk nanoparticles. Silk fibers degummed with different media at different time intervals were investigated for the degumming loss and were characterized using Ffourier transform infrared (FTIR), differential scanning calorimetry (DSC), x-ray diffraction (XRD), and scanning electron microscopy (SEM). Maximum degumming was observed when the fibers were treated with sodium carbonate for 60 min. SEM and atomic force microscopy (AFM) images of RSF solution showed aggregation of silk globules resulting in formation of solvated macrochains and giving it an appearance of island-like morphology. Blank silk nanoparticles prepared from the RSF solution showed a smooth and spherical surface devoid of any adhesion using SEM, AFM, and transmission electron microscopy (TEM). The prepared silk nanoparticles may further be explored for loading drug entities and targeting.     

Mulberry non-engineered silk gland protein vis-à-vis silk cocoon protein engineered by silkworms as biomaterial matrices

Silk fibroin from silk gland of Bombyx mori 5th instar larvae was utilized to fabricate films, which may find possible applications as two-dimensional matrices for tissue engineering. Bombyx mori cocoon fibroin is well characterized as potential biomaterial by virtue of its good mechanical strength, water stability, thermal properties, surface roughness and biocompatibility. The present study aims to characterize the biophysical, thermal, mechanical, rheological, swelling properties along with spectroscopic analysis, surface morphology and biocompatibility of the silk gland fibroin films compared with cocoon fibroin. Fibroin solutions showed increased turbidity and shear thinning at higher concentration. The films after methanol treatment swelled moderately and were less hydrophilic compared to the untreated. The spectroscopic analysis of the films illustrated the presence of various amide peaks and conformational transition from random coil to beta sheet on methanol treatment. X-ray diffraction studies also confirmed the secondary structure. Thermogravimetric analysis showed distinct weight loss of the films. The films were mechanically stronger and AFM studies showed surfaces were rougher on methanol treatment. The matrices were biocompatible and supported L929 mouse fibroblast cell growth and proliferation. The results substantiate the silk gland fibroin films as potential biomaterial matrices.     

Fabrication of novel biofibers by coating silk fibroin with chitosan impregnated with silver nanoparticles

Nanoparticle based agents often applied as coatings on biomaterials have shown promise in delivering the improved sterility against variety of microbes. In the present study, silk fibers (SF) were coated with chitosan impregnated with silver nanoparticles (Ag–C–SF). These Ag–C–SF fibers were characterized using scanning electron microscopy (SEM) with energy dispersive X-ray analysis, atomic force microscopy (AFM), Infra Red spectroscopy, Thermogravimetric Analysis and Microbiological assay techniques. AFM studies have confirmed the nano sized silver particles in chitosan solution; SEM pictures have exhibited the coating of chitosan along with silver nanoparticles on the silk fibroin. The modified fibers have also shown anti-microbial activity and improved thermal stability. The Ag–C–SF fibers may be explored as wound dressing and tendon reconstruction material in future.     

SILK.IT project: Silk Italian Technology for industrial biomanufacturing

Silk fibroin is a natural protein that constitutes the core fibre of the silkworm cocoon. Recently, a great deal of attention is being paid to the use of the regenerated silk fibroin (RSF) solution that can be obtained by water-processing of the cocoon fibre. Indeed, thanks to its chemical and physical properties it has been shown that substrates obtained by RSF can be used as biomaterials for several biomedical and technological applications. In this context, silk fibroin could be exploited as raw material that can become a platform for eco-sustainable manufacturing.The project SILK.IT, coordinated by the CNR-ISOF, within the framework of the Flagship Project “Factory of the Future”, a research programme approved by the Interministerial Committee for Economic Planning (CIPE), aims to support this assumption by developing methods and protocols to use this silk fibroin as new material for advanced biotechnologies and sustainable manufacturing.The main goal of the project is to establish and control the whole-chain underpinning the silk fibroin-based technology, and to promote its up-scaling from the laboratory to industrial scale, targeting bio-photonics for biomedical application.The recent results obtained by the project are herein reported as key building block towards proposed achievement to establish a sustainable and process-controlled silk-based bio-manufacturing of the future.     

Techno‐mechanical properties of cocoon,raw silk and filament of two mulberry silkworm (Bombyx mori L.) strains

Natural silk is considered as queen of textile due to its superior traits. This study was conducted to assess technical properties of cocoon, and raw silk and mechanical properties of silk filament produced by two mulberry silkworm strains. The Chinese strain (205PO) produced dry cocoon of 0.61±0.04 g with raw silk of 0.30±0.02 g and the Japanese strain (J101) produced dry cocoon of 0.49±0.01 g with raw silk of 0.23±0.00 g. The single filament length of 205PO and J101 was 1203.1±20.42 m cocoon^?1 and 1082.3±48.95 m cocoon^?1, respectively. The filament was finer in the Japanese silkworm strain (1.91±0.06 denier) compared to the Chinese silkworm strain (2.26±0.15 denier). The filament tenacity, tenacity rupture and strain of J101 was 6.24 %, 24.62 % and 4.42 % greater compared to 205PO. The tensile strength of 205PO was 11.82 % greater compared to J101. The filament diameter was 22.01±0.42 μm and 21.98±0.15 μm of 205PO and J101, respectively. Based on these findings, it is recommended that silkworm strains with superior techno‐mechanical properties may be included in breeding programmes for enhancing the quality of silk textile.     

Silk fibroin protein from mulberry and non-mulberry silkworms: cytotoxicity, biocompatibility and kinetics of L929 murine fibroblast adhesion

Silks fibers and films fabricated from fibroin protein of domesticated mulberry silkworm cocoon have been traditionally utilized as sutures in surgery and recently as biomaterial films respectively. Here, we explore the possibility of application of silk fibroin protein from non-mulberry silkworm cocoon as a potential biomaterial aid. In terms of direct inflammatory potential, fibroin proteins from Antheraea mylitta and Bombyx mori are immunologically inert and invoke minimal immune response. Stimulation of murine peritoneal macrophages and RAW 264.7 murine macrophages by these fibroin proteins both in solution and in the form of films assayed in terms of nitric oxide and TNFalpha production showed comparable stimulation as in collagen. Kinetics of adhesion of L929 murine fibroblasts, for biocompatibility evaluation, monitored every 4 h from seeding and studied over a period of 24 h, reveal A. mylitta fibroin film to be a better substrate in terms of rapid and easier cellularization. Cell viability studies by MTT assay and flow cytometric analyses indicate the ability of fibroin matrices to support cell growth and proliferation comparable to collagen for long-term culture. This matrix may have potential to serve in those injuries where rapid cellularization is essential.     

Preparation and characterization of regenerated fiber from the aqueous solution of Bombyx mori cocoon silk fibroin

The regenerated silk fibers with high strength and high biodegradability were prepared from the aqueous solution of Bombyx mori silk fibroin from cocoons with wet spinning method. Although the tensile strength of the regenerated silk fibroin fiber, 210 MPa is still half of the strength of native silk fiber, the diameter of the fiber is about 100 μm which is suitable for monofilament of suture together with high biodegradability. The high concentration (30%, w/v) of the aqueous solution of the silk fibroin which corresponds to the high concentration in the middle silkgland of silkworm was obtained. This was performed by adjusting the pH of the aqueous solution to 10.4 which corresponds to pK_a value of the OH group of Tyr residues in the silk fibroin. The mixed solvent, methanol/acetic acid (7:3 in volume ratio) was used as coagulant solvent for preparing the regenerated fiber. The structural change of silk fibroin fiber by stretching was monitored with both ¹³C solid state NMR and X-ray diffraction methods, indicating that the high strength of the fiber is related with the long-range orientation of the silk fibroin chain with β-sheet structure.     

Effects of High Magneto-Gravitational Environment on Silkworm Embryogenesis

The objective of this research was to observe whether silkworm embryos can survive in a high magneto-gravitational environment (HMGE) and what significant phenotype changes can be produced. The hatching rate, hatching time, life span, growth velocity and cocoon weight of silkworm were measured after silkworm embryos were exposed to HMGE (0 g, 12 T; 1 g, 16 T; and 2 g, 12 T) for a period of time. Compared with the control group, 0 g exposure resulted in a lower hatching rate and a shorter life span. Statistically insignificant morphological changes had been observed for larvae growth velocity, incidence of abnormal markings and weight of cocoons. These results suggest that the effect of HMGE on silkworm embryogenesis is not lethal. Bio-effects of silkworm embryogenesis at 0 g in a HMGE were similar with those of space flight. The hatching time, life span and hatching rates of silkworm may be potential phenotype markers related to exposure in a weightless environment.     

Tissue response and biodegradation of composite scaffolds prepared from Thai silk fibroin,gelatin and hydroxyapatite

This work aimed to investigate tissue responses and biodegradation, both in vitro and in vivo, of four types of Bombyx mori Thai silk fibroin based-scaffolds. Thai silk fibroin (SF), conjugated gelatin/Thai silk fibroin (CGSF), hydroxyapatite/Thai silk fibroin (SF4), and hydroxyapatite/conjugated gelatin/Thai silk fibroin (CGSF4) scaffolds were fabricated using salt-porogen leaching, dehydrothermal/chemical crosslinking and an alternate soaking technique for mineralization. In vitro biodegradation in collagenase showed that CGSF scaffolds had the slowest biodegradability, due to the double crosslinking by dehydrothermal and chemical treatments. The hydroxyapatite deposited from alternate soaking separated from the surface of the protein scaffolds when immersed in collagenase. From in vivo biodegradation studies, all scaffolds could still be observed after 12 weeks of implantation in subcutaneous tissue of Wistar rats and also following ISO10993-6: Biological evaluation of medical devices. At 2 and 4 weeks of implantation the four types of Thai silk fibroin based-scaffolds were classified as “non-irritant” to “slight-irritant”, compared to Gelfoam^® (control samples). These natural Thai silk fibroin-based scaffolds may provide suitable biomaterials for clinical applications.     

原子力显微镜下蚕丝及蜘蛛丝的微观结构

利用原子力显微镜研究了蚕丝丝素、蜘蛛牵引丝及其内外层包卵丝的微观结构.研究表明,丝素和包卵丝纤维的纵向表面都有成丝过程中液态丝蛋白流动而形成的清晰的构槽和条纹,在低速下自然分泌的牵引丝的表面皮层相对比较细腻,而垂直下落蜘蛛在高速下分泌的牵引丝具有和丝素纤维比较相似的微观结构特征.这些丝纤维的断面内都分布有大量微细的原纤,形状基本为圆形,其中三种蜘蛛丝的微纤维直径相似,而丝素纤维内的微纤维要粗得多.     

Green synthesis and characterisation of antioxidant-tagged gold nanoparticle (X-GNP) and studies on its potent antimicrobial activity

Alternanthera philoxeroides (Mart.), a weed, mainly from tropical origin and easily available worldwide. People used to eat it as a food mainly in South Africa. In our previous report we have thoroughly characterise several important phenolics, monoterpene and phenylpropane from methanol soluble fraction of Alternenthera leaves (fraction X) and also reported their α-glucosidase inhibitory, antimicrobial and antioxidant activities. All these isolated natural compounds are well characterised and widely studied. In our present study we try to use this beneficial fraction (named fraction X) in green synthesis of gold nanoparticles (X-GNP). We also try to explore the beneficial aspects of green synthesis in comparison with commonly used chemical synthesis method (GNP) in context with their antimicrobial activity. UV/Vis spectroscopy, DLS, Zeta potential, FT-IR, EDAX and other microscopic techniques namely: SEM, AFM were used to characterise the synthesised nanoparticles. Different important microbial strains were used to evaluate the antimicrobial activity of prepared nanoparticles. Overall the studies suggest successful synthesis of green nanoparticles (X-GNP) and also showed the improvement in antimicrobial activity of X-GNP nanoparticles.     

Morphology and structure of silkworm cocoons

Silkworm cocoons are natural polymer fibre composites made from silk fibres and sericin binder. While silk is an interesting natural material per se, an understanding of the role of silk within one of its main functional applications in silkworm cocoons will provide inspiration and tools for the design of new artificial silk composites. Here, we describe in some detail the structure and morphology of the cocoons of 27 different species of silkworm. While cocoon morphology can be described very broadly as a nonwoven fibre composite, we demonstrate a diversity of structural features such as: the number and connectivity of layers through the cocoon wall thickness, the amount and distribution of sericin binder, the diameter and packing density of the silk fibres, the degree of orientation of the nonwoven structure, the distribution of larger holes within that structure, and the presence of calcium oxalate crystals.     

Impact of different silkworm dietary supplements on its silk performance

This study aims to evaluate the effect of silkworm larva (Bombyx mori) diet supplementation with two amino acids (threonine and valine) on the cocoon production and on the structural and mechanical properties of the silk produced. Negligible morphological differences were observed in the silk fiber threads from silkworm larvae supplemented with the tested amino acids. Higher production (yield) of silk was obtained using threonine in the diet of the silkworm. The treatments with threonine have increased the limit of proportionality, tensile strength, toughness, and maximum deformation of the thread of silk fibers. No significant increment in these properties was observed due to the increase in the threonine content. The treatments with valine led to lower increase in tensile strength and toughness. The real density of the silk has decreased with the use of supplements. The present study contributes to engineering of advanced silk materials, which should be attractive candidates for multipurpose applications.     

EDC/NHS交联改性再生桑蚕丝素纳米纤维

用静电纺丝技术制备了再生桑蚕丝素纳米纤维,并用1-(3-二甲基氨基丙基)-3-乙基碳化二亚胺(EDC)和N-羟基丁二酰亚胺(NHS)进行交联改性,考察了交联改性前后,桑蚕丝素纳米纤维微观形貌及聚集态结构等的变化,采用扫描电镜(SEM)、X射线衍射(XRD)及红外光谱(FT-IR)等测试方法对纳米纤维进行表征。研究结果表明,经EDC/NHS交联改性后,纤维直径由250 nm增加到320 nm,纤维表面变粗糙,且纤维发生弯曲变形;丝素的结构以Silk II为主,并含有部分无规卷曲或Silk I构象;桑蚕丝纳米纤维的力学性能和亲水性有所提高,且交联改性后的纳米纤维具有良好的细胞相容性。     

Photocatalytic degradation of ciprofloxacin pollutant and in-vitro cytotoxic activity of gold nanoparticles using seed extract of Abrus precatorius

Synthesis of nanoparticles from plant extract is a very simple reliable, rapid, affordable and un-hazardous technique. In the present exploration, gold nanoparticles (Au NPs) were rapidly synthesized using seed extract of Abrus precatorius. The prepared nanomaterials were systematically proved by FTIR, UV–Visible, Powder-XRD, SEM, EDAX and TEM analyses. The formation of Au NPs was primarily identified by the colour change from yellow to purple within 5 min and which showed a surface Plasmon peak around 535 nm. The dimension of the gold nanoparticles was found to be in the range of 10–20 nm. The particles were poly-dispersed with a triangular, spherical and hexagonal shape. The gold nanoparticles were used to degrade the antibiotic drug ciprofloxacin (CIP) with an efficiency of above 95%. Moreover, the bio-synthesized gold nanoparticles had been assessed for their in-vitro cytotoxic activity on human breast cancer cells (MCF-7) lines. The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to evaluate the Inhibitory Concentration (IC₅₀) value. The IC₅₀ value is 37?±?1.5 µg/mL. In future, this can be used for breast cancer therapy.

     

β-glucosidase assisted biosynthesis of gold nanoparticles: A green chemistry approach

Biogenic gold nanoparticles using medically important phytochemical (β-glucosidase) are demonstrated herein. The reduction capabilities of phytochemical and their ability to stabilize the new genre gold nanoparticles, were characterized using UV-visible, FT-IR, HR-TEM and XRD measurements. This report also focuses the newly formed gold nanoparticles application on promoting the defensive mechanism of silkworm Bombyx mori. The results highlight the possibility of green pathways to produce functionalized gold nanoparticles of medicinal significance.     

Silver nanoparticle production by Rhizopus stolonifer and its antibacterial activity against extended spectrum β-lactamase producing (ESBL) strains of Enterobacteriaceae

This report focuses on the synthesis of silver nanoparticles using the fungus, Rhizopus stolonifer and its antimicrobial activity. Research in nanotechnology highlights the possibility of green chemistry pathways to produce technologically important nanomaterials. Characterization of newly synthesized silver nanoparticles was made by UV-visible absorption spectroscopy, scanning electron microscope (SEM), transmission electron microscope (TEM), Fourier transform infrared (FTIR) spectroscopy and atomic force microscope (AFM). TEM micrograph revealed the formation of spherical nanoparticles with size ranging between 3 and 20 nm. The biosynthesized silver nanoparticles (AgNPs) showed excellent antibacterial activity against ESBL-strains which includes E. coli, Proteus. sp. and Klebsiella sp.     

Magnetic Properties of Nanostructured Co and Ni Synthesized by Modified NaBH4 Reduction Route

Nanomaterials based on Co and Ni are technologically important because of their potential technological applications in recording media, catalysis, drug delivery systems, and so on. Recent research interests lie on the synthesis of Co and Ni nanomaterials by chemical synthesis, characterizations and studying for their interesting magnetic properties. In this investigation, we have focused on the synthesis of cobalt and nickel nanoparticles (NPs) in aqueous medium at ambient conditions by sodium borohydride reduction route. We have successfully stabilized the nanospheres comprising of Co and Ni by using polyethylene glycol (PEG) as capping agent. The Co and Ni nanomaterials were exhaustively characterized by x-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and magnetic measurements. The phase purity and crystallite sizes were ascertained by using powder XRD method. Co and Ni NPs crystallize in face centered cubic (fcc) structure with lattice parameters (a) equal to 3.54 Å and 3.52 Å, respectively. The XRD lines were broad and indicate the fine particle nature of the materials. The estimated crystallite sizes were found to be 42 and 29 nm for Co and Ni, respectively. SEM micrograph studies show the particle sizes to be 80 and 70 nm, whereas TEM studies confirm the sizes to be 47 and 65 nm for Co and Ni, respectively. The electron micrograph studies indicate the appearance of agglomerates of the nanoparticles consisting of several crystallites. The specific magnetization versus field characteristics of Co and Ni nanoparticles shows the signature of the size and surface effects. The values of saturation magnetizations are found to be 122 and 47 emu/g, whereas the coercivity values are 111 Oe and 84 Oe for Co and Ni, respectively. In summary, we have synthesized high moment Co and Ni nanostructured materials with reduced coercivities, which may be useful for soft magnetic applications.     

A novel electrospun silk fibroin/hydroxyapatite hybrid nanofibers

A novel electrospinning of silk fibroin/hydroxyapatite hybrid nanofibers with different composition ratios was performed with methanoic acid as a spinning solvent. The silk fibroin/hydroxyapatite hybrids containing up to 30% hydroxyapatite nanoparticles could be electrospun into the continuous fibrous structure. The electrospun silk fibroin/hydroxyapatite hybrid nanofibers showed bigger diameter and wider diameter distribution than pure silk fibroin nanofibers, and the average diameter gradually increased from 95 to 582 nm. At the same time, the secondary structure of silk fibroin/hydroxyapatite nanofibers was characterized by X-ray diffraction, Fourier transform infrared analysis, and DSC measurement. Comparing with the pure silk fibroin nanofibers, the crystal structure of silk fibroin was mainly amorphous structure in the hybrid nanofibers. X-ray diffraction results demonstrated the hydroxyapatite crystalline nature remained as evidenced from the diffraction planes (002), (211), (300), and (202) of the hydroxyapatite crystallites, which was also confirmed by Fourier transform infrared analysis. The thermal behavior of hybrid nanofibers exhibited the endothermic peak of moisture evaporation ranging from 86 to 113 °C, and the degradation peak at 286 °C appeared. The SF/HAp nanofibers mats containing 30% HAp nanoparticles showed higher breaking tenacity and extension at break for 1.1688 ± 0.0398 MPa and 6.55 ± 1.95%, respectively. Therefore, the electrospun silk fibroin/hydroxyapatite hybrid nanofibers should be provided potentially useful options for the fabrication of biomaterial scaffolds for bone tissue engineering.     

Silver nanoparticles incorporated electrospun silk fibers

We present a simple and mass-producible method of incorporating silver nanoparticles on the surface of electrospun silk non-woven membranes for the fabrication of antimicrobial wound dressings. Nanofibrous silk membranes with fiber diameters of 460 +/- 40 nm were electrospun from an aqueous Bombyx mori fibroin solution. The electrospun membranes incorporating silver nanoparticles were prepared by dipping the membranes in aqueous silver nitrate (AgNO3) solution (0.5 or 1.0 wt%) followed by photoreduction. Field emission scanning and transmission electron microscopy showed that silver nanoparticles were generated on the electrospun silk fibroin nanofibers as well as inside them. The interaction between the silver nanoparticles and amide groups in the silk fibroin molecules was characterized using X-ray photoelectron spectroscopy.