Chemical Modification of Silk Proteins: Current Status and Future Prospects

Silk extracted from the cocoon of silkworm has been used as textile materials for thousands of years.Recently,silk has been redefined as a protein-based biomaterial with great potential in biomedical applications owing to its excellent mechanical properties,biocompatibility,and biodegradability.With the advances in silk processing technologies,a broad range of intriguing silk-based functional biomaterials have been made and applied for various biomedical uses.However,most of these materials are based on natural silk proteins without chemical modification,leading to limited control of properties and functions(e.g.,biodegradability and bioactivity).A chemical toolbox for modifying the silk proteins is required to achieve versatile silk-based materials with precisely designed properties or functions for different applications.Furthermore,inspired by the traditional fine chemical industry based on synthetic chemistry,developing silk-based fine chemicals with special functions can significantly extend the applications of silk materials,particularly in biomedical fields.This review summarizes the recent progress on chemical modification of silk proteins,focusing on the methodologies and applications.We also discuss the challenges and opportunities of these chemically modified silk proteins.

Production of Functional Materials Derived from Regenerated Silk Fibroin by Utilizing 3D Printing and Biomimetic Enzyme-induced Mineralization

Critical-sized bone defects, commonly encountered in clinical orthopedic surgery, present a significant challenge. One of the promising solutions is to prepare synthetic bone substitute materials with precise structural control, mechanical compatibility, and enhanced osteogenic induction performance, nevertheless the successful preparation of such materials remains difficult. In this study, a two-step technique,integrating an extrusion-based printing process with biomimetic mineralization induced by alkaline phosphatase(ALP), was developed. Initially,a pre-cured hydrogel of regenerated silk fibroin(RSF) with a small quantity of hydroxypropyl cellulose(HPC) and ALP was prepared through heating the mixed aqueous solution. This pre-cured hydrogel demonstrated thixotropic property and could be directly extruded into predetermined structures through a 3D-printer. Subsequently, the 3D-printed RSF-based materials with ALP underwent biomimetic in situ mineralization in calcium glycerophosphate(Ca-GP) mineralizing solution, utilizing the polymer chains of RSF as templates and ALP as a trigger for cleaving phosphate bonds of Ca-GP. The resulting 3D-printed RSF-mineral composites including hydrogel and sponge possessed adjustable compression modulus of megapascal grade and variable hydroxyapatite content, which could be controlled by manipulating the duration of the mineralization process.Moreover, these 3D-printed RSF-mineral composites demonstrated non-cytotoxicity towards rat bone marrow mesenchymal stem cells. Therefore, they may hold great potential for applications involving the replacement of tissues characterized by osteoinductivity and intricate structures.

Improving the hydrostability of zeolitic imidazolate framework coatings using a facile silk fibroin protein modification method

Zeolitic imidazolate frameworks(ZIFs)are an important subclass of metal-organic frameworks(MOFs)with zeolite-type topology,which can be fabricated under ambient synthesis conditions.However,the applications of ZIFs are commonly limited due to the weak hydrostability of their metal–ligand coordination bonds,particularly under humid and aqueous conditions.In this work,as an example,the hydrolysis behaviours of ZIF-L with a special focus on ZIF-L coatings were tested at aqueous conditions with a wide range of pHs to systematically study and fundamentally understand their structural stability and degradation mechanism.Pristine ZIF-L powder and ZIF-L coatings were severely damaged after only 24 h in aqueous media.Interestingly,the ZIF-L coatings showed two distinct hydrolyzation pathways regardless of pH conditions,exhibiting either a ring-shaped etching or unfolding behaviours.While the ZIF-L powders were hydrolyzed almost identically across all pH conditions.With this new understanding,a facile silk fibroin(SF)protein modification method was developed to enhance the hydrostability of ZIF-L coatings in aqueous media.The effect of protein concentration on surface coating was systemically studied.ZIF-L coating retained its surface morphology after soaking in water and demonstrated switchable super wetting properties and superior separation performance for oil/water mixture.As a result,the quick SF protein modification significantly enhanced the stability of ZIF-L coatings under various pHs,while retaining their switchable wetting property and excellent separation performance.

Assessment of Sericin Biosorbent for Selective Dye Removal

The silk sericin is the main residue in silk production and it is found to be a low cost and efficient bio-sorbent. In this study, sericin was characterized with various techniques including SEM （scanning electron micro- scope）, XRD, N2 physisorption, FTIR （Fourier transformed infrared spectroscopy） and XPS （X-ray photoelectron spectroscopy）. The nitrogen content of sericin was ca. 8.5 mmol.g-1 according to elemental analysis. Dye adsorption by sericin biosorbent was investigated with the acid yellow （AY）, methylene blue （MB） and copper （II） phthalocyanine-3,4＇4＂4＇＂-tetrasulfonic acid （CuPc） dyes from water. Sericin displayed large capacity for AY andCuPc adsorption with adsorption capacities of respectively 3.1 and 0.35 mmol.g-1, but it did not adsorbed methyl- ene blue dye. This selectivity is due to the basicity of amide groups in seriein biosorbents.

Construction of sustainable, colored and multifunctional protein silk fabric using biomass riboflavin sodium phosphate

Riboflavin sodium phosphate has been confirmed as a promising biomass product derived from natural plants.In this paper,a novel method of dyeing and multifunctional modification of silk fabric by impregnation with riboflavin sodium phosphate was proposed,such that protein silk fabric can be endowed with bright yellow color and multi-functionality.The results of this paper confirmed that the pH and concentration of riboflavin sodium phosphate solution are critical factors for dyeing and multifunctional modification.Attractively,the photochromic performance was one of the most distinctive features of the modified silk fabric,and the dyed silk fabric turned into fluorescent green from original yellow under 365 nm ultraviolet lamp.Furthermore,the modified silk fabric exhibited good antibacterial properties with a high inhibition rate of 92%for Escherichia coli.Besides,the flame retardancy of silk fabric was significantly improved after modification.The damaged length of modified silk fabric with 40%owf riboflavin sodium phosphate was lower than 10.4 cm and passed the B1 classification.As revealed by the result of this paper,riboflavin sodium phosphate is sufficiently effective in serving as an eco-friendly multifunctional agent for strengthening the add-value of silk textiles.

Analysis of transcriptomes of three orb-web spider species reveals gene profiles involved in silk and toxin

As an ancient arthropod with a history of 390 million years, spiders evolved numerous morphological forms resulting from adaptation to different environments. The venom and silk of spiders, which have promising commercial applications in agriculture, medicine and engineering fields, are of special interests to researchers. However, little is known about their genomic components, which hinders not only understanding spider biology but also utilizing their valuable genes. Here we report on deep sequenced and de novo assembled transcriptomes of three orb-web spider species, Gasteracantha arcuata, Nasoonaria sinensis and Gasteracantha hasselti which are distributed in tropical forests of south China. With Illumina paired-end RNA-seq technology, 54 871, 101 855 and 75 455 unigenes for the three spider species were obtained, respectively, among which 9 300, 10 001 and 10 494 unique genes are annotated, respectively. From these annotated unigenes, we comprehensively analyzed silk and toxin gene components and structures for the three spider species. Our study provides valuable transcriptome data for three spider species which previously lacked any genetic/genomic data. The results have laid the first fundamental genomic basis for exploiting gene resources from these spiders.

An atomistic model of silk protein network for studying the effect of pre-stretching on the mechanical performances of silks

Silk protein builds one of the strongest natural fibers based on its complex nanocomposite structures.However,the mechanical performance of silk protein,related to its molecular structure and packing is still elusive.In this study,we constructed an atomistic silk protein network model,which reproduces the extensive connection topology of silk protein with structure details of theβ-sheet crystallites and amorphous domains.With the silk protein network model,we investigated the structure evolution and stress distribution of silk protein under external loading.We found a pre-stretching treatment during the spinning process can improve the strength of silk protein.This treatment improves the properties of silk protein network,i.e.,increases the number of nodes and bridges,makes the nodes distributed homogeneously,and induces the bridges in the network well aligned to the loading direction,which is of great benefit to the mechanical performances of silk protein.Our study not only provides a realized atomistic model for silk protein network that well represents the structures and deformations of silk proteins under loading,but also gains deep insights into the mechanism how the pre-loading on silk proteins during spinning improves the mechanical properties of silk fibers.

The Silk Textile Embedded in Silk Fibroin Composite: Preparation and Properties

Silk reinforced silk-fibroin-based composites were prepared by embedding of silk textile into regenerated silk fibroin （RSF） matrix. The breaking stress and breaking strain of the composites were found 37.7 MPa and 71.1% respectively at （95 ：t： 5）% RH. Morphological analysis was carried out to observe fracture behavior of the samples. The in vitro biodegradation test showed that the composite degraded slowly and lost 70% weight at the end of 168 h. Moreover, compared with RSF pure film, the composite kept strength and toughness much longer time. In conclusion, this composite has the potential for more accurate cytology research and biomedical tests in the future.

Engineering homologous platelet-rich plasma,platelet-rich plasma-derived exosomes,and mesenchymal stem cell-derived exosomes-based dual-crosslinked hydrogels as bioactive diabetic wound dressings

The management of diabetic wounds remains a critical therapeutic challenge. Platelet-rich plasma (PRP) gel, PRP-derived exosomes (PRP-Exos), and mesenchymal stem cell-derived exosomes (MSC-Exos) have demonstrated therapeutic potential in wound treatment. Unfortunately, their poor mechanical properties, the short half-lives of growth factors (GFs), and the burst release of GFs and exosomes have limited their clinical applications. Furthermore, proteases in diabetic wounds degrade GFs, which hampers wound repair. Silk fibroin is an enzyme-immobilization biomaterial that could protect GFs from proteases. Herein, we developed novel dual-crosslinked hydrogels based on silk protein (SP) (sericin and fibroin), including SP@PRP, SP@MSC-Exos, and SP@PRP-Exos, to promote diabetic wound healing synergistically. SP@PRP was prepared from PRP and SP using calcium gluconate/thrombin as agonist, while SP@PRP-Exos and SP@MSC-Exos were derived from exosomes and SP with genipin as crosslinker. SP provided improved mechanical properties and enabled the sustained release of GFs and exosomes, thereby overcoming the limitations of PRP and exosomes in wound healing. The dual-crosslinked hydrogels displayed shear-induced thinning, self-healing, and eradication of microbial biofilms in a bone-mimicking environment. In vivo, the dual-crosslinked hydrogels contributed to faster diabetic wound healing than PRP and SP by upregulating GFs expression, down-regulating matrix metalloproteinase-9 expression, and by promoting an anti-NETotic effect, angiogenesis, and re-epithelialization. Hence, these dual-crosslinked hydrogels have the potential to be translated into a new generation of diabetic wound dressings.

Actively separated microneedle patch for sustained-release of growth hormone to treat growth hormone deficiency

Growth hormone deficiency(GHD)has become a serious healthcare burden,and presents a huge impact on the physical and mental health of patients.Here,we developed an actively separated microneedle patch(PAA/NaHCO_(3)^(-)Silk MN)based on silk protein for sustained release of recombinant human growth hormone(rhGH).Silk protein,as a friendly carrier material for proteins,could be constructed in mild full-water conditions and ensure the activity of rhGH.After manually pressing PAA/NaHCO_(3)^(-)Silk MN patch to skin for 1 min,active separation is achieved by absorbing the interstitial fluid(ISF)to trigger HCO_(3)^(-)in the active backing layer to produce carbon dioxide gas(CO_(2)).In rats,the MN patch could maintain the sustained release of rhGH for more than 7 days,and produce similar effects as daily subcutaneous(S.C.)injections of rhGH in promoting height and weight with well tolerated.Moreover,the PAA/NaHCO_(3)^(-)Silk MN patch with the potential of painless self-administration,does not require cold chain transportation and storage possess great economic benefits.Overall,the PAA/NaHCO_(3)^(-)Silk MN patch can significantly improve patient compliance and increase the availability of drugs,meet current unmet clinical needs,improve clinical treatment effects of GHD patients.

Effect of quercetin on chondrocyte phenotype and extracellular matrix expression

Due to the poor repair ability of cartilage tissue,regenerative medicine still faces great challenges in the repair of large articular cartilage defects.Quercetin is widely applied as a traditional Chinese medicine in tissue regeneration including liver,bone and skin tissues.However,the evidence for its effects and internal mechanisms for cartilage regeneration are limited.In the present study,the effects of quercetin on chondrocyte function were systematically evaluated by CCK8 assay,PCR assay,cartilaginous matrix staining assays,immunofluorescence assay,and western blotting.The results showed that quercetin significantly up-regulated the expression of chondrogenesis genes and stimulated the secretion of GAG(glycosaminoglycan)through activating the ERK,P38 and AKT signalling pathways in a dose-dependent manner.Furthermore,in vivo experiments revealed that quercetin-loaded silk protein scaffolds dramatically stimulated the formation of new cartilage-like tissue with higher histological scores in rat femoral cartilage defects.These data suggest that quercetin can effectively stimulate chondrogenesis in vitro and in vivo,demonstrating the potential application of quercetin in the regeneration of cartilage defects.