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Silk fibers (SF) reeled from silkworms are constituted by natural proteins, and their characteristic structural features render them applicable as materials for textiles and packaging. Modification of SF with functional materials can facilitate their applications in additional areas. In this work, the preparation of functional SF embedded with carbon dots (CD) is reported through the direct feeding of a CD-modified diet to silkworms. Fluorescent and mechanically robust SF are obtained from silkworms (Bombyx mori) that are fed on CDs synthesized from the Morus rubra variant of mulberry leaves (MB-CDs). MB-CDs are introduced to silkworms from the third instar by spraying them on the silkworm feed, the mulberry leaves. MB-CDs are synthesized hydrothermally without adding surface passivating agents and are observed to have a quantum yield of 22%. With sizes of ≈4 nm, MB-CDs exhibited blue fluorescence, and they can be used as efficient fluorophores to detect Dopamine (DA) up to the limit of 4.39 nM. The nanostructures and physical characteristics of SF weren't altered when the SF are infused with MB-CDs. Also, a novel DA sensing application based on fluorescence with the MB-CD incorporated SF is demonstrated., (© 2023 Wiley-VCH GmbH.)

Hydrogels, being capable of mimicking the extracellular matrix composition of tissues, are greatly used as artificial matrices in tissue engineering applications. In this study, the generation of horseradish peroxidase (HRP)-crosslinked silk fibroin (SF) hydrogels, using calcium peroxide as oxidizer is reported. The proposed fast forming calcium-containing SF hydrogels spontaneously undergo SF conformational changes from random coil to β-sheet during time, exhibiting ionic, and pH stimuli responsiveness. In vitro response shows calcium-containing SF hydrogels' encapsulation properties and their ability to promote SaOs-2 tumor cells death after 10 days of culturing, upon complete β-sheet conformation transition. Calcium-containing SF hydrogels' angiogenic potential investigated in an in ovo chick chorioallantoic membrane (CAM) assay, show a high number of converging blood vessels as compared to the negative control, although no endothelial cells infiltration is observed. The in vivo response evaluated in subcutaneous implantation in CD1 and nude NCD1 mice shows that calcium-containing SF hydrogels are stable up to 6 weeks after implantation. However, an increased number of dead cells are also present in the surrounding tissue. The results suggest the potential of calcium-containing SF hydrogels to be used as novel in situ therapeutics for bone cancer treatment applications, particularly to osteosarcoma., (© 2021 Wiley-VCH GmbH.)

Polymeric microcapsules have begun to attract significant interest in biomedical fields. As the interactions between cells and materials are influenced by both cell type and elasticity, silk-based microcapsules are synthesized with desirable mechanical features using layer-by-layer assembly and then the uptake of these microcapsules by BeWo b30 placental cells is investigated. Cellular uptake is enhanced with increasing of elastic modulus of the silk-based microcapsules. More importantly, the distinct microvilli of these cells behaves in a diverse manner when exposed to microcapsules with different mechanical features, including grabbing (rigidity) or random touching (soft) behavior; these factors affect the final uptake. Inspired by oocyte pickup, the grabbing behavior of the microvilli may provide valuable information with which to elucidate the specific characteristics of uptake between cells and man-made particles, particularly in the reproductive system., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The present study focuses on the alkaline phosphatase (ALP) mediated formation of apatitic minerals on porous silk fibroin protein (SFP) scaffolds. Porous SFP scaffolds impregnated with different concentrations of ALP are homogeneously mineralized under physiological conditions. The mineral structure is apatite while the structures differ as a function of the ALP concentration. Cellular adhesion, proliferation, and colonization of osteogenic MC3T3 cells improve on the mineralized SFP scaffolds. These findings suggest a simple process to generate mineralized scaffolds that can be used to enhanced bone tissue engineering-related utility., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The effect of some sterilization methods (autoclaving and ethanol treatments) on the degradation rate and mechanical properties of two types of porous silk scaffolds (aqueous- and hexafluoroisopropanol-derived) is evaluated. Changes in secondary structure, crystal size, and supramolecular features of silk fibroin, resulting from sterilization, are tracked to elucidate molecular level effects on protease XIV enzymatic degradation and compressive mechanical properties. The structural features and pore sizes of the silk scaffolds remain intact after both sterilization processes. Autoclave sterilization dramatically reduce the degradation rate of the silk scaffolds in response to protease XIV and significantly increase mechanical properties, in contrast to scaffolds sterilized with 70% ethanol. Higher β-sheet content and larger crystal size are observed after autoclaving, unlike in response to 70% ethanol sterilization, based on examination of Fourier transform (FT) IR spectroscopy and wide-angle X-ray scattering (WAXS). In addition, thermal analysis finds supramolecular features within silk fibroin amorphous regions, including the glass transition temperature (Tg ), heat capacity of glass transition (ΔCp-Tg ), and thermal gravimetric degradability. Such supramolecular level changes are related to the shift in enzymatic degradation and mechanical properties due to autoclaving versus treatment with 70% EtOH. The changes in supramolecular organization in amorphous regions can retard enzyme diffusion through the glassy regions of the silk matrix or/and hinder binding of enzymes, while also stiffening these matrices., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mulberry silk fibroin is a widely used biomaterial and recent work on non-mulberry silk fibroin also suggests it may have similar uses. We expect silk fibroin from the only domesticated non-mulberry eri silkworm, Samia ricini, to possess useful properties as a biomaterial. Eri silk gland fibroin is a heterodimeric protein of approximately 450 kDa. Cytocompatibility evaluation with fibroblasts and osteoblast-like cells shows good cell attachment, viability and proliferation. The matrices, which have high thermal stability and good swellability, are also haemocompatible. Eri silk production is cost effective as no agronomic practices are required for their host plant cultivation. This fibroin provide new opportunities as an alternative natural functional biomaterial in various biomedical applications., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recombinant 6mer + BSP protein, combining six repeats of the consensus sequence for Nephila clavipes dragline (6mer) and bone sialoprotein sequence (BSP), shows good support for cell viability and induces the nucleation of hydroxyapatite and tricalcium phosphate during osteoblast in vitro culture. The present study is conducted to characterize this bioengineered protein-based biomaterial further for in vivo behavior related to biocompatibility. 6mer + BSP protein films are implanted in subcutaneous pouches in the back of mice and responses are evaluated by flow cytometry and histology. The results show no major differences between the inflammatory responses induced by 6mer + BSP films and the responses observed for the controls. Thus, this new chimeric protein could represent an alternative for bone regeneration applications., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A significant challenge remains to protect protein drugs from inactivation during production, storage, and use. In the present study, the stabilization and release of horseradish peroxidase (HRP) in silk films was investigated. Water-insoluble silk films were prepared under mild aqueous conditions, maintaining the activity of the entrapped enzyme. Depending on film processing and post-processing conditions, HRP retained more than 90% of the initial activity at 4 degrees C, room temperature and 37 degrees C over two months. The stability of protein drugs in silk films is attributed to intermolecular interactions between the silk and the enzymes, based on Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The unique structural feature of silk molecules, periodic hydrophobic-hydrophilic domains, enabled strong interactions with proteins. The entrapped protein was present in two states, untrapped active and trapped inactive forms. The ratio between the two forms varied according to processing conditions. Proteolytic degradation and dissolution of the silk films resulted in the release of the bound enzyme which was otherwise not released by diffusion; enzyme recovered full activity upon release. There was a linear relationship between silk degradation/dissolution and the release of entrapped enzyme. Modifying the secondary structure of the silk matrix and the interactions with the non-crystalline domains resulted in control of the film degradation or dissolution rate, and therefore the release rate of the entrapped enzyme. Based on the above results, silk materials are an intriguing carrier for proteins in terms of both retention of activity and controllable release kinetics from the films.