Biosynthesis of insulin-silk fibroin nanoparticles conjugates and in vitro evaluation of a drug delivery system

Silk fibroin derived from Bombyx mori is a biomacromolecular protein with outstanding biocompatibility. When it was dissolved in highly concentrated CaCl₂ solution and then the mixture of the protein and salt was subjected to desalting treatments for long time in flowing water, the resulting liquid silk was water-soluble polypeptides with different molecular masses, ranging from 8 to 70 kDa. When the liquid silk was introduced rapidly into acetone, silk protein nanoparticles with a range of 40–120 nm in diameter could be obtained. The crystalline silk nanoparticles could be conjugated covalently with insulin alone with cross-linking reagent glutaraldehyde. In vitro properties of the insulin-silk fibroin nanoparticles (Ins-SFN) bioconjugates were determined by Enzyme-Linked Immunosorbent Assay (ELISA). The optimal conditions for the biosynthesis of Ins-SFN bioconjugates were investigated. The Ins-SFN constructs obtained by 8 h of covalent cross-linking with 0.7% cross-linking reagent and the proportion of insulin and SFN being 30 IU: 15 mg showed much higher recoveries (90–115%). When insulin was coupled covalently with silk nanoparticles, the resistance of the modified insulin to trypsin digestion and in vitro stability in human serum were greatly enhanced as compared with insulin alone. The results in human serum indicated that the half-life in vitro of the biosynthesized Ins-SFN derivatives was about 2.5 times more than that of native insulin. Therefore, the silk protein nanoparticles have the potential values for being studied and developed as a new bioconjugate for enzyme/polypeptide drug delivery system.     

Silk-mediated synthesis and modification of photoluminescent ZnO nanoparticles

In this article, a bio-inspired silk-mediated method was established to produce natural material-modified photoluminescent zinc oxide nanoparticles (nano-ZnO). Silk fibroin fibers were employed as the reactive substrates to synthesize nano-ZnO, and silk fibroins (SF) were taken as the biocompatible stabilizers to modify dispersed nano-ZnO. As-prepared nano-ZnO were mainly hexagonal phase particles with diameter around 13 nm. The resulting nano-ZnO/SF hybrids displayed orange emission and good biocompatibility in aqueous system.     

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.     

High-affinity integration of hydroxyapatite nanoparticles with chemically modified silk fibroin

Hydroxyapatite (HA)-based nanocomposites were prepared by a co-precipitation method with silk fibroin (SF) serving as organic matrix. Silk fibroin was chemically modified with an alkali solution or an enzyme attempting to improve the interface between the mineral and the organic matrix. The influences of the alkali and enzyme pretreatments on microstructure and physicochemical properties of HA–SF composite were examined and compared. The results reveal that both the two kinds of pretreatments facilitate the formation of highly ordered three-dimensional porous network throughout the composites, increase the microhardness of the composite, and promote the preferential growth of HA crystallites along c-axis. Among all the as-prepared samples, the composite containing the enzyme pretreated SF shows desirable hierarchical microstructure with higher degree of organization and more uniform pore size distribution. Due to the enzyme pretreatment, HA crystallites undergo obvious changes in morphology from rod-like to␣whisker-like and in crystal growth towards more apparent epitaxy along c-axis. The alkali pretreatment induces the stronger chemical interactions between HA and SF and thus to strengthen the inorganic–organic interfacial adhesion. The newly developed HA–SF composites are expected to be attractive biomedical materials for bone repair and remodeling.     

Light can transform the secondary structure of silk protein

Fibroin is the main component of silk and is expected to be used as a novel functional material in medicine and bioelectronics. The main secondary structures of this protein are of the random-coil and the β-sheet types. In this study, we carried out laser-induced transformation of the secondary structure, from the random-coil type to the β-sheettype, in solid fibroin films. We prepared two types of fibroin films with the random-coil structure. One is a fibroin film doped with a dye as a photosensitizer with a small amount (1 wt %), and the other is a neat fibroin film. The former was excited at 532 nm and the latter was excited at 266 nm. Irradiations were carried out with fluences much lower than each ablation threshold. The excitation of the dye at 532 nm did not affect the secondary structure of the random-coil type. By contrast, 266-nm laser irradiation, which excites tryptophan (an amino-acid residue) involved in fibroin, created the β-sheetdomain in the film. The structural transformation was revealed by infrared absorption spectroscopy and atomic force microscopy. Received: 1 August 2001 / Accepted: 2 August 2001 / Published online: 2 October 2001     

磁共振技术在丝素蛋白结构与功能研究中的应用

丝素蛋白纤维因其优异的力学性能和良好的生物相容性而得到广泛关注. 人们通过对丝素蛋白二级结构的构象转变及其诱导因素的研究，试图阐明蚕吐丝机理、相关蛋白结构与功能的关系，为人工合成性能优良的丝纤维材料以及认识生命过程提供有益指导和帮助. 磁共振技术是研究丝素蛋白结构最有效的方法之一. 我们课题组多年来运用核磁共振（NMR）的方法研究了桑蚕丝素蛋白的构象及环境对其的影响因素，如pH、金属离子（K⁺、Na⁺、Ca²⁺、Cu²⁺、Zn²⁺）等，并运用电子自旋顺磁共振（EPR）方法研究了金属Cu²⁺离子与丝素蛋白的相互作用，试图揭示蛋白质构象转变与金属离子影响的内在联系. 另外，我们还发展了一种广义二维核磁共振相关技术，有效缩短了传统二维核磁共振的实验时间. 本文将综述这些年来我们所做的相关工作.     

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.     

Topochemistry,optical anisotropy and FT-IR microspectroscopy of the cocoon of Lithurgus chrysurus (Hymenoptera,Megachilidae)

A previous study has not revealed the participation of a mucous component in the cocoon wall of the solitary bee, Lithurgus chrysurus, differing from the cocoon structure reported for many other bee species. However, uncertainty remains, because only the median and rear zones of this cocoon type have thus far been analyzed. Here, we studied the front zone of this cocoon, searching its components and their organization, to fill this knowledge gap. Topochemical assays, polarization microscopy and Fourier transform-infrared (FT-IR) microspectroscopy were used to study cross sections from L. chrysurus cocoon. Three main layers differing in structural organization were found to compose the cocoon wall. Silk fibroins were assumed to constitute the filamentous threads of the inner and outer layers and the laminar structure of the intermediate layer. Deduced from its topochemical properties and FT-IR spectral signature, a foamy material containing mucin glycoproteins and carboxylated acid glycosaminoglycans was found in the intermediate layer. FT-IR analysis using a Savitzky-Golay 2nd-derivative and absence of linear dichroism and birefringence phenomena suggest that a random-coil secondary structure predominates in the foam component. Co-existence of α-helical and β-sheet conformations is also hypothesized for the fibroin component of this cocoon. It is thus concluded that in addition to fibroin elements, a mucous component, likely contributed by a Malpighian tubule secretion, integrates the composition of the front zone of the cocoon wall of L. chrysurus. In addition, the FT-IR analysis of the inner layer silk of this cocoon suggests significant differences in comparison to the silk fibroins of the silkworm, and some minor spectral differences in comparison to published data on the honeybee silk, with respect to protein secondary structure.     

Effect of gamma irradiation on synthesis and characterization of bio-nanocomposite SF/Ag nanoparticles

This study describes the synthesis of silver nanoparticles (AgNPs) using aqueous silk fibroin (SF) solution obtained from Bombyx mori silk under gamma radiation environment. The obtained AgNPs were characterized using UV–visible (UV–Vis) spectroscopy, X-ray diffraction (XRD) measurements, dynamic light scattering experiment (DLS) and transmission electron microscope (TEM) images. The UV–Vis absorption spectra of the samples confirmed the formation of AgNPs by showing surface plasmon resonance (SPR) band in the range of (= 428–435?nm. The XRD study revealed metal silver with the face-centered cubic (FCC) crystal structure. DLS measurements showed the dose-dependent average size of the AgNPs. TEM images showed formed AgNPs are nearly spherical in shape with smooth edges. From this study, it was found that the increasing radiation dose increases the rate of reduction and decreases the particle size. The size of the AgNPs can be tuned by controlling the radiation dose.