Fabrication And Mechanism Analysis Of Silk Fibers With High Strength And Toughness By Directly Feeding Silkworm Additives

As a traditional bio-resource in China,silkworm silk has been developed and used for thousands of years.Silkworm silk,a typical natural biological protein material,has gained considerable attention because of its good mechanical properties,and favorable biocompatibility and biodegradability.It is thus applicable as a textile as well as biomedical material,optoelectronic devices,flexible electronic materials,biosensors,etc.However,artificial synthetic fiber can imitate the appearance or characteristics of natural fiber with the development of chemical technology.The increasing artificial synthetic fiber products in the market have a certain impact on the traditional silk and its products.Although the comprehensive properties of silkworm silks are favorable,they still needs to be improved comparing with spider silk?another animal protein fiber?which has quite high strength and extensibility.Many studies have focused on improving the performance of silkworm silk fibers to make them rival or even exceed spider silks,as spider silk cannot be easily prepared by domestic raising like silkworm silk.To achieve this goal,currently four major methods,including genetic modification,post treatment,forced reeling and feeding silkworm with modified diets,have been developed in fabrication of silk fiber with enhanced mechanical properties or multifunction.It is of particular interest that feeding silkworm larvae with a series of additives?e.g.,organic dyes,nanoparticles?NPs?,as well as some special organics?can improve the mechanical properties of silk fiber and enrich its functions.As the feeding method maintains the feature of natural silk fibers and can easily get the in-situ modified silk fibers,it is a green and sustainable in-situ modified approach to produce high-performance and functional silk fibers on a large scale.In this study,we chose the fifth instar silkworm larvae as bioreactor and different types of materials as silkworm additives to investigate the interaction between additives and silk proteins as well as the biological interaction between silkworm larvae and additives by chemical and biological testing techniques.In addition,the relationship between the structure and properties of silkworm silk and silkworm additives were systematically studied from the view of materials science.The major achievements and conclusions were summarized as follows:1.The biological effects of feeding metal nanoparticles on silkworm.Two commonly used nanomaterials?Cu NPs and Ag NPs?were chose as additives for fifth instar silkworm larvae to explore the biological effects of additives on silkworms.The distributions of the metallic element in their tissues were investigated using ICP-MS and the results showed that Cu or Ag NPs were absorbed into all the silkworm larvae tissues and eventually spun into silk fibers,although most of the metallic NPs were excreted daily.Toxicity of NPs on silkworm were evaluated by silkworm larvae weights,survival rates,quanlity of cocoons as well as the histopathological microstructures.Comparing with Ag NPs,Cu NPs had a more negative influence on the physiological behaviors of the silkworms and cocooning,such as a lower growth rate,reduced feed efficiency,the construction of lightweight cocoon shells.However,the cocoon shell ratio of cocoons were not significantly changed after NPs were added.Histopathological observations showed that the midgut and malpighian tubule of silkworm were obviously damaged after feeding NPs.Biochemical indicators including T-AOC level,SOD activity,CAT activity,GSH content and MDA content were tested to investigate the toxic mechanism caused by Cu NPs or Ag NPs.These test results showed that T-AOC level,SOD activity,CAT activity and GSH content in the hemolymph and different organizations of silkworm have different degrees of reduction whereas MDA content in the hemolymph and organizations of silkworm?especially in the midgut?showed a significant increase.Among them,significant changes in these biochemical indicators were mainly observed in epidermises,midgut and hemolymph.Comparing with Ag NPs,more oxidative damages were found in these tissues treated by Cu NPs.It suggested that the balance between oxidation and antioxidation system of silkworm tissues was broken and the anti-oxidation defense system was destroyed after feeding NPs.It inhibited the activity of antioxidant enzymes in the tissues?especially in epidermises,midgut and hemolymph?,resulting in a large amount of reactive oxygen species accumulated in these tissues.This triggers the lipid peroxidation and causes serious damages to silkworm tissues.Therefore,silkworm larvae showed stunted growth or death after feeding with NPs.Digital gene expression?DGE?tag was used to analyze the gene expression profile of the posterior silk gland of silkworms after fed with Cu or Ag NPs.Compared with the control group,Cu NPs treated silkworms showed 102 differentially expressed genes,including 77 upregulated genes and 25 downregulated genes.Ag NPs treated silkworms showed 114 differentially expressed genes,including 59 upregulated genes and 55downregulated genes.Among these differentially expressed genes,the genes related to keratin,spiral protein and germicidal peptide precursor were upregulated in Cu NPs group while the genes related to heatshock protein,flocculating protein and sulfurtransferase were upregulated in Ag NPs group.There are only 15 common differentially expressed genes in these two groups,which are related to signal transduction,protein synthesis and neuropeptide receptor.Those upregulated genes play an important role in the stimulation of additives,synthesis and secretion of silk protein.Pathway mapping using the Kyoto Encyclopedia of Genes and Genomes?KEGG?showed that metabolic pathway-related genes and endocytosis pathway-related genes were the most significantly enriched.It suggested that Cu or Ag NPs affect the metabolism system of silkworm.In addition,the endocytosis may be one of the pathway for the interaction between NPs and protein.2.The effects of feeding metal nanoparticles on silk gland protein and silk fibers.In order to elucidate the influence mechanism of Cu NPs and Ag NPs on silk fiber,sodium dodecyl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?,fluorescence spectroscopy?FS?as well as circular dichroism?CD?were used.The structural effects of Cu NPs and Ag NPs on silk gland protein from silkworm larvae feeding with NPs were investigated.Meanwhile,NPs were used to induce silk gland protein in vitro to analyze the interaction between NPs and proteins without the effects of the organism.The results indicated that middle silk gland protein at 43 kDa from Cu NPs group and Ag NPs group were more highly expressed comparing with the control group;posterior silk gland protein at 60 kDa in those two modified groups were less expressed.From the results of FS and CD,we can conclude that NPs change the microenvironment of luminescent groups in silk protein such as tyrosine and serine,leading to fluorescence quenching.However,NPs induced the transformation from?-helix and random coils to?-sheet structures in modified silk gland.The in vitro results showed that the combination of Cu NPs or Ag NPs with silk gland protein slightly inhibits the?-sheet conformation transition.The inconsistency of interaction between NPs and silk gland proteins in vivo and in vitro is due to the interference of microenvironment in silkworm body.The morphology,amino acid content,secondary structure and properties of the silkworm silk were investigated using scanning electron microscope?SEM?,Fourier transform infrared?FTIR?spectroscopy,amino acid analyses,thermogravimetric analysis?TGA?and quasi-static tensile tests.The results indicated that the morphology and basic secondary structures of silk fibers had not been virtually changed due to the addition of NPs whereas NPs hindered the conformation transition from random coil/?-helixes to?-sheets of silk fibroin.The amino acid content were changed with the NPs added,resulting in larger SC/LC amino acid chain ratios.Furthermore,metallic NPs in the study were proved to enhance the thermal stability and mechanical properties of the silk fibers with a coupling competitive mechanism.Tensile strength and toughness of silk fibers from Cu NPs group were increased by 31%and 26%respectively compared to control silk,and the two indices were 19%and 13%respectively for silk fibers from Ag NPs group.3.Research on the effects and mechanism of metallic ions and amino acid substances on secondary structure and mechanical properties of silk fibersFor more suitable material for growth of silkworm and modification of silk properties,eight types of materials including nano materials?Cu and CaCO₃?,metallic ions(Ca²⁺and Cu²⁺)and amino acid substances?serine,tyrosine,sericin amino acids,and fibroin amino acids?were used as additives in silkworm diet to obtain in-situ modified silk fibers.The impacts of these additives on the metallic element contents,conformational transitions,and physical properties of silk fibers were systematically investigated to reveal the reinforcement mechanism of these different materials.Our findings indicate that:the copper element-contained or calcium element-contained additives were absorbed by silkworm larvae through their diets and eventually spun into silk fibers;the additives without copper components reduced the copper content of silk fibers.More importantly,the content of potassium in silkworm silk modified by amino acid substances increased by about 10 times compared with control silk.The results from FTIR and XRD both indicated that feeding silkworm with tyrosine or fibroin amino acids can induce the transformation of silk proteins from?-helices and random coils to?-sheet structures.The?-sheet contents of tyrosine-and fibroin amino acids-modified silk fibers were about 58%and 63%,respectively,which were markedly higher than that of the control group?45%?.Meanwhile,compared with the control silk that showed an overall crystallinity of 36%,increases in crystallinity were observed in silk fibers modified by tyrosine?40%?and fibroin amino acids?49%?.However,the?-sheet content and crystallinity values in the other six modified fibers were relatively lower than that of the control fibers.The results from TGA and mechanical properties test showed that Cu NPs,serine and sericin amino acids improved the thermal stability of silk.Meanwhile,tensile strength were significantly improved by feeding Cu NPs,serine,tyrosine and fibroin amino acids,and the best tensile strength happened on the silk fiber modified by tyrosine,1.4 times as much as that of the control silk.Moreover,Young's modulus of silk from sericin amino acid group increased by 27%compared with control silk while the index decreased in silk fiber modified by metallic ions.More importantly,feeding silkworm with tyrosine or fibroin amino acids can obtain high-toughness silk fibers,which was 1.4 times and 1.5times as much as that of the control silk,respectively.The results above suggest that the enhanced mechanical properties of silk fibers modified by nanoparticles can be mainly attributed to the high performance of the materials themselves;amino acid-modified diets can change the potassium content and increase the crystallinity of silk fibers.The role of exogenous metallic ions is similar to that of amino acid,but they were unfavorable for silk fibroin conformation and resulted in weaker stiffness of silk fibers.4.Fabrication of luminescent silk fiber with superior mechanical property and highly stable fluorescence by feeding and its applicationIn combination with the enhanced mechanism of nanomaterials and the fabrication and application status of functional fluorescent silkworm silk,we produced two kinds of fluorescent silk fibers with superior mechanical properties,high stable fluorescence and good biocompatibility via directly feeding silkworm with Graphene quantum dots?GQDs?or CdSe/ZnS quantum dots?CQDs?modified diets.RhB were used as the comparison group.In contrast to our expectations,80%GQDs or 89%CQDs were found in the fibroin,which was much higher than the content of RhB,70%.After 30 days of water soaking,70-80%QDs retained in the fibroin while only 23%RhB can be found,indicating the good fluorescent stability of the QDs-containing silk fibers in water.In addition,the tensile strength increased by 1.27-fold up and 1.40-fold up for GQDs group and CQDs group respectively compared with the control group.Tensile toughness for those two groups increased by 1.48-fold up and 1.97-fold up respectively.Further,MTT and flow cytometry results reveal that GQDs and CQDs modified silk fibers exhibit lower cytotoxicity and better cytocompatibility comparing with RhB modified silk fibers.QDs modified silk fibers with the superior mechanical properties and fluorescent stability as well as biocompatibility has the promising applications in fluorescent clothing,anti-counterfeiting marks,cancer detection,bio-imaging and even the optical sensing.In summary,this paper systematically studied the bio-interaction between silkworm and additives and the relationship between silk fibers and additives from the perspectives of multidisciplinary such as material,chemical and biological system.This study could provide the useful guidance for the development and application of silk fibers modified by direct feeding additives from macroscopic appearances to in-depth mechanism analysis.