REVIEW ON FABRICATION AND APPLICATION OF REGENERATED BOMBYX MORI SILK FIBROIN MATERIALS

processability, superior biocompatibility, controllable biodegradation, and versatile functionalization. Tremendous effort has been made to fabricate silk fi broin into various promising materials with controlled structural and functional characteristics for advanced utilities in a multitude of biomedical applications, fl exible optics, electronics devices, and fi ltration systems. Herein, reverse engineered silk fi broin extraction methods are reviewed, recent advances in extraction techniques are discussed. Fabrication methods of silk fi broin materials in various formats are also addressed in detail; in particular, progress in new fabrication technologies is presented. Attractive applications of silk fi broin-based materials are then summarized and highlighted. The challenges faced by current approaches in production of silk fi broin-based materials and future directions acquired for pushing these favorable materials further toward above mentioned applications are further elaborated.

Conventionally, sericin is removed by a process known as degumming with only silk fi broin left for industrial applications because it has improved mechanical properties, luster, and softhandling than the raw silk [11]. Due to its hierarchical structure and versatility, the utilization of B. mori silk fi broin has extended from the textile industry to various high-tech applications in the form of multiple regenerated B. mori silk fi broin material formats. Inspired by the ability of B. mori silkworms to synthesize and store liquid silk, researchers have attempted to use the topdown approach to reverse B. mori silk fi broin fi lament into solution then to solidify into versatile forms such as fi bers, particles, fi lms, hydrogels, and three dimensional (3D) materials for multiple applications such as wearable electronic devices, photonic devices, water ultrafi ltration systems, biosensors, drug delivery systems, and tissue engineering [12,13]. The purpose of this review is to provide an overview of recent progress in the development of regenerated B. mori silk fi broin materials for diverse applications. Reverse engineered silk fi broin extraction methods are fi rst introduced, and then recent advances in extraction techniques are discussed. Fabrication methods of regenerated silk fi broin materials in various formats are also addressed in detail, and in particular, progress in new fabrication technologies is presented. Attractive applications of either pure or composite regenerated silk fi broin materials are then summarized and highlighted. The challenges faced by current approaches in the production of regenerated silk fi broin materials and future directions acquired for pushing these favorable materials further toward the above-mentioned applications are further elaborated.

Reverse engineered B. mori silk fi broin extraction
Despite various silk fi broin-based materials regeneration methods, a two-step silk fi broin extraction process including degumming and dissolving is essential.

Degumming of raw silk
Silk degumming is the process by which the gummy layer of sericin, which binds two fi broin fi laments, is removed by breaking peptide bonds present between sericin and fi broin through thermochemical treatment of cocoons or raw silk. It has been reported that sericin can cause allergic reactions and infections in humans and inhibit the premature conversion of soluble silk (silk I) into gelated and β-sheet silk conformations [14]. Therefore, it is necessary to eliminate sericin and other impurities for better utilization of the silk. Conventional degumming is based on using boiling water or aqueous solutions containing soap, alkaline, acid, enzyme, or amine to hydrolyze the sericin (Figure 2) [15]. Comparative analysis of various degumming methods is provided in Table 1.
Water degumming is a green processing method and the cheapest one involving no use of chemicals. However, high temperature, long and repeated processing times, and pressure are always required for effi cient removal of sericin. Besides, it is reported that water degumming has the possibility of causing silk fi broin damage at such high temperature for long time, and the problem of incomplete degumming. Thus, this process is not commercially adopted. Water degumming Degumming of silk using dilute acid, such as tartaric acid, lactic acid, oxalic acid, citric acid, succinic acid, malonic acid, trichloroacetic acid, monochloroacetic acid, glacial acetic acid, and sulfuric acid, is an approach that has long been practiced. Effective acid degumming occurs in the pH range of 1.5-2. These dilute acids specifi cally perform a hydrolytic attack on sericin protein to break peptide bonds between aspartic acid and glutamic acid. Acid degumming provides very fast degumming rate and avoids the use of high temperature. However acid degumming is used less due to the fact that alkaline solution is safer for fi broin than acids [22,23].
Degumming of silk by enzymes such as trypsin, papain, and bacterial enzymes is also a method that has long been practiced. The mechanism of enzymatic degumming is that the proteolytic enzymes cleave the peptide and amide linkages of sericin and convert them into amino acid. Different enzymes show effective degumming at different pH. Compare this with nonspecifi c hydrolytic reactions of other methods: studies have indicated that proteolytic enzymes react only at specifi c sites. Since degumming by this method is generally carried out at comparatively low temperatures ranging from 37°C to 60°C under mildly alkaline or acid conditions, enzymatically degummed silk fi bers are less damaged with a higher degree of surface whiteness and soft handle compared with soap-or alkali-treated silk fi bers. The disadvantage of this method is that hydrophobic impurities such as natural wax, twisting oil, and sericin cannot be completely removed due to the mild processing conditions. Moreover, it is unsuitable for large-scale use due to its low effi ciency [24,25].
Organic amines such as methylamine, ethylamine, diethylamine, and triethylamine have also been investigated as alternatives degumming agents to optimize the degumming conditions. The mechanism of amine degumming is similar to that of alkalis, since sericin removal from silk fi bers by this method is associated with the fact that amines ionize in water to give hydroxyl (OH) ions. Reports have shown that under optimized conditions amine degumming shows a degumming effect comparable with Marseilles soap degumming but that was less susceptible to the hardness of water. The disadvantage of this method is that gradual removal of sericin leads to a low degumming rate [26,27].
Until now, water treatment has been used to soften and partially dissolve sericin to enable the silk fi lament unwinding in silk reeling in textile industry. Na 2 CO 3 is the most highly preferred is commonly used to soften and partially dissolve sericin by soaking cocoons into water with a temperature of 50-60℃ to enable the silk fi lament unwinding in silk reeling in the textile industry [16].
Soap degumming involving treating silk with a slightly alkaline soap solution is one of the most commonly used method. Various sodium-based soap, such as sodium stearate, sodium myristate, sodium laurate, sodium arachidate, sodium oleate, sodium ricinoleate, and sodium caprylate, based on distinct oils such as olive oil, palm oil, coconut oil, laurel oil, canola oil, palm kernel oil, and tallow oil have been employed for soap degumming purposes. The removal of sericin by soap solutions is due to alkali formation upon soap hydrolysis, which forms chemical bonds with sericin and turns it into soluble soda salt. Therefore, soaps with greater hydrolysis capacity perform more effective degumming. The degumming effect of soapbased treatment is also infl uenced by parameters such as temperature, degumming time, liquor ratio, and pH of solution. Generally, the quantity of soap, degumming temperature, degumming time, liquor ratio, and pH required with the soap solution may approximately take the values of 20%-30% soap to the weight of material, 90-95℃,1.5-2 h, 30-40:1, 9.7-10.5, respectively. Though the soap degumming method has advantages of mild processing, avoidance of over-degumming, and increasing the softness, whiteness, absorbency, and luster properties of the resultant fi broin fi bers, the load of soap in the solution is high, and it is susceptible to hard water. In order to avoid metallic complex deposits and resulting stains, the water should be properly softened [17][18][19].
Alkali degumming is another most commonly used method both in industry and laboratory since it's benefi cial both on economic and quality grounds. Various alkalis, including sodium carbonate (Na 2 CO 3 ), sodium bicarbonate (NaHCO 3 ), sodium hydroxide (NaOH), potassium carbonate (KCO 3  strongly alkaline electrolyzed water (SAEW) with a pH of 11.5 as a degumming agent by electrolyzing tap water with a laboratory-made combined water electrolyzer. The hardness of SAEW was found to be 30% less than that of the tap water, whereas the concentrations of Na + in SAEW were 18% higher than those in the tap water. Their results showed that there was no evident difference among the surface properties of silk fi broin degummed by SAEW, Na 2 CO 3 and neutral soap degumming methods ( Figure 3) [44]. Wang et al. performed pressurized steam treatment on raw silk to remove sericin. It was found that steam treatment with lower pressure (0.14 MPa) and long residence time (90 min) generates no adverse effects on the physicochemical properties of the degummed silk fi bers [45].

Dissolving of degummed silk
Dissolving of the degummed silk is the next step followed by degumming of silk. Either aqueous or nonaqueous silk fi broin solution can be obtained from which a range of silk fi broinbased materials in versatile formats can be fabricated [46,47]. The dissolving of silk fi broin in inorganic salt systems is due to hydrogen bond disruption in the crystalline structure (ordered, hydrophobic, and high-density β sheet region). It is worth noting that the dissolved silk fi broin salt solution requires extensive dialysis and concentration before the regenerated silk fi broin is suitable for use. The concentration process is normally done by dialysis against an aqueous polyethylene glycol solution (20 wt%) to extract water [15, 46, 47]. Sometimes freezedrying followed by redissolving at required concentration is needed. Thus, the whole dissolving, dialysis, concentration, and freeze-drying processes for extraction of regenerated silk fi broin are time-consuming [4]. In addition, the regenerated silk  Nonaqueous silk fibroin solution primarily used for producing regenerated fibers and films via wet spinning, dry spinning, and electrospinning. The most commonly used solvents for artificial spinning are hexafluoro-2-propanol (HFIP), hexafluoroacetone (HFA), formic acid, and formic acid/CaCl 2. [47, 49, 50]. These solvents first cause decomposition of the hydrogen bonds and then break down the peptide bonds. However, these solvents are strongly corrosive, toxic, and volatile. And the obtained silk fibroin solution is not conducive to preservation. Ecofriendly solvents such as N-Methylmorpholine N-oxide (NMMO) and ionic liquids such as 1-Allyl-3-methylimidazolium chloride (AMIMCl), 1-Butyl-3-methylimidazolium chloride (BMIMCl), 1-Butyl-3-methylimidazole acetate (BMIMAc), 1-Ethyl-3-methylimidazolium chloride (EMIMCl), 1-Butyl-3-methylimidazolium bromide (BMIMBr), 1-Butyl-3methylimidazolium iodide (BMIMI), etc. have also been explored for dissolving. Compared with the organic solvents or inorganic salt solvents, dissolution in ionic liquids has the advantages of simple, rapid, and greener preparation processes; stable solutions; elimination of the use of toxic organic volatile liquids; and convenient recycling. However, dissolution in ionic liquid needs high temperatures, which can lead to the degradation of silk protein [46, 47, 50, 51].
Recently, Wang et al. proposed a green and environmentfriendly method to enhance efficiency of aqueous silk fibroin solution preparation. In particular, they used excess acetone to extract concentrated silk fibroin-LiBr solution for dialysis. The required dialysis time can be reduced by half using this method. It was found that the stability, peptide chains, and secondary structure of regenerated silk fibroin are minimally changed. In addition, the resulting acetone and salt in wastewater can be easily separated and recycled for silk fibroin regeneration [52]. Yet, it can be seen that the extraction of regenerated silk fibroin with only mild degradation and stable β-sheet conformation in large scale by cost effective and easy methods is still a problem.

B. mori silk fibroin materials fabrication
After researchers successfully obtain the silk solution, the regeneration of silk fibroin into novel material formats such as fibers, particles, films, hydrogels, and 3D materials with different morphologies can be done, according to the type of end-application ( Figure 4).
In comparison with natural silk fiber, regenerated silk fibers can be prepared by wet spinning, dry spinning, and electrospinning. Generally, silk fibroin solution used for artificial spinning is primarily nonaqueous silk fibroin dissolved with organic solvents or ionic liquids because of the better rheological properties of the solution and better evaporation of the solvent [46,47]. In wet spinning, the silk protein solution is extruded through a spinneret directly into a coagulation bath that initiates solidification into fibers via precipitation. In contrast, dry spinning solidification of the fiber occurs due to the evaporation of a volatile solvent [59]. Electrospinning is a simple and useful technique for producing nano/micro fi bers possessing large surface areas. By these artifi cial spinning processes, the diameter, morphology, and mechanical properties of the regenerated silk fi broin fi bers obtained can be conveniently modifi ed. Moreover, the regenerated silk fi broin fi bers can be endowed with diverse optical, electrical, and biofunctional properties by the addition of various functional fi llers during fabrication [60]. For example, Ma et al. fabricated photochromic regenerated silk fi broin/Tungsten trioxide nanoparticles hybrid fi bers via wet spinning process [60]. Mohsen et al. fabricated short staple microfi bers based on chitosan or silk fi broin via the wet spinning technique to adsorb hexavalent chromium from aqueous solution [61]. Mostafavi et al. fabricated vitamin D-loaded silk fi broin/polycaprolactone (SF/PCL) nanofi brous scaffolds for bone tissue engineering applications [62]. Besides these conventional processes, straining fl ow spinning recently appears as a versatile and robust technique that allows effi cient spinning of regenerated silk fi bers. Geometrical and hydrodynamic processing parameters can be integrated into this technique, which allows spinning under a much wider range of chemistries [63]. Liquid exfoliation, which directly exfoliates silk nanofi brils from degummed silk fi bers, is another newly developed method that can preserve the native structural elements and features of silk fi bers with the aim to rearrange them at the nanofi bril level for enhanced properties [64,65].
Silk fi broin particles can be fabricated by either the top-down method or the bottom-up method. The top-down method involves simply chopping, grinding, and crushing the SF degummed fi bers into smaller aggregates. This method generally results in big nanoparticle aggregates with a wide size range and needs a long time and specifi c apparatus [8,46]. The bottom-up method uses the silk fi broin solution as the starting material. Broad bottom up-methods such as selfassembly, freeze-drying, and grinding, freeze-thawing, spraydrying, jet-breaking, electro-spraying, microfl uidic technique, dissolution, and salting out are applied to produce silk fi broin particles [8,66]. The self-assembly method takes advantage of silk fi broin self-assembly behavior ruled by hydrophilic and hydrophobic chain interactions. For example, Xiao et al. fabricated regenerated silk fi broin microsphere-embedded Fe 3 O 4 magnetic nanoparticles for immobilization of zymolyase through a controllable ethanol-induced interface self-assembly. The prepared microsphere exhibited high immobilization effi ciency [67]. Spray-drying, jet-breaking, and electro-spraying are similar technologies, which involve the breaking down of the aqueous silk fi broin fl ow into small droplets by force [8,68]. Dissolution represents the most common method for obtaining silk fi broin particles. It is based on the reduction of the silk fi broin chain solubility in the presence of organic solvents such as acetone, ethanol, dimethyl sulfoxide (DMSO), and methanol, leading to phase separation [69,70]. Salting out is another approach employed for the production of silk fi broin particles. The formation of particles results from the increased hydrophobic interaction between the SF protein chains due to the dehydration effect of the added salt ions. The manipulation of the solution pH has been found to have a profound effect on the silk secondary structure, zeta potential, as well as salting out efficiency [59].
Silk fibroin films can be easily prepared by solution casting, spin-coating, or layer-by-layer deposition methods [2,14]. Water insoluble and flexible silk fibroin films can be fabricated by adjustment of β-sheet content in the films via controlled drying, water annealing, stretching, and organic solvents treatment [56,62]. Wang et al. fabricated silk fibroin films with tunable ductility and porosity by adjusting the protein self-assembly process through combinations with glycerol and polyethylene glycol 400 and regulating the film-casting temperature. Among various conditions screened, the composite film with a mass ratio of silk fibroin/polyethylene glycol 400/glycerol of 10:5:3 prepared at 4°C exhibited remarkable ductility with a tensile strength of 2.7 ± 0.2 MPa and an elongation at the break of 164.24 ± 24.20% [71]. Silk films with micro/nano-scale patterned morphology can be prepared by the nanolithography technology including soft-lithography, photolithography, ion beam lithography, and nanoimprinting. Micro/nano-scale patterned silk fibroin films have the potential being explored for guided cell growth and as optical storage medium [72,73]. Softlithography involves casting the aqueous silk fibroin solution on nanostructured surfaces made from elastomeric molds such as polydimethylsiloxane (PDMS) [74]. Ion beam lithography uses an accelerated beam of ions to create nanostructures on cure silk fibroin films [75]. Photolithography is an efficient and costeffective technique that uses light to define patterns on cure silk fibroin films [76].
Silk fibroin hydrogels can be prepared through sol-gel transition of fibroin aqueous solution triggered by physical or chemical processes, where a molecular network forms by controlling the formation of β-sheet structures as crosslinks for silk [14,59]. Physically crosslinked silk fibroin hydrogels can be produced by temperature control, application of shear force, ultrasonication, and electrical current, etc. The underlying mechanism of this process is the self-assembly of proteins via enhanced hydrophobic interactions. Chemically crosslinked silk fibroin hydrogels can be produced by pH control; addition of precipitating agents such as concentrated salt solutions, organic solvents, surface agents, large polymeric precipitating agents, and small neutral additives; chemical stabilization; and chemical modification. Generally, a lower pH and a higher incubation temperature facilitate increased kinetics toward the sol-gel transition [77,78]. In addition to the above-mentioned methods, Hasturk et al. recently fabricated silk-gelatin hydrogel scaffold with tunable gelation kinetics, mechanical properties and bioactivity for cell culture and encapsulation via enzymatic crosslinking [79]. Marín et al. synthesized composite hydrogel of regenerated silk fibroin incorporating gold nanoparticles (AuNPs), silver nanoparticles (AgNPs), or iron oxide nanoparticles (IONPs) using bisphosphonate-decorated nanoparticles (NPs) to prevent their aggregation and enzymeassisted cross-linking method to control the formation of the silk fibroin composite hydrogels in the presence of the NPs [80]. Confined alignment is another new method used to fabricate silk fibroin hydrogel with controlled molecular alignment and degree of crystallinity. This method enables the design and engineering of biopolymer-based materials with controlled hierarchical structure at multiple length scales and tunable properties [81].
Various forms of silk fibroin-based 3D materials such as porous scaffolds, sponges, foams, tubes, rods, and screws can be fabricated by several most widely adopt techniques including salt-leaching, gas foaming, freeze-drying, and electrospinning. In the salt-leaching method, salts are added to the silk fibroin solution, the mixture is then placed in a mold. After the mixture solution turns into gel, the salt is removed by immersing the mixture in water to obtain the salt-free 3D porous scaffolds. However, one major issue with this method is that the leaching of salt particles can lead to poor interconnection and abnormal pore shape. Gas foaming is a kind of method to create an interconnected pore structure with high porosity by rapid pressure relief around the liquefied polymer. Pore structure and porosity of the fabricated constructs can be tuned by varying the amount of gas in polymer. Freeze-drying is an important means to fabricate silk fibroin-based porous sponge scaffolds. The pore size and microstructures can be effectively controlled by adjusting the freezing temperature or silk fibroin concentration. Moreover, aligned scaffolds can be obtained by controlling the frozen conditions [2,3,[82][83][84][85]. Besides these conventionally used methods, Guo et al. recently fabricated silk fibroin-based bulk materials including bars, rods, plates, tubes with caps, and screws with tunable mechanical properties via thermal processing for medical device applications. Specifically, amorphous silk nanomaterials reconstructed from the LiBr-silk fibroin solution were first produced, which were then processed into robust structural materials by hot pressing [86]. The method of 3D printing is also new, allowing the formation of silk fibroinbased 3D materials with biologically relevant structures in defined geometries with microscale resolution that are difficult or impossible to manufacture with conventional fabrication techniques. Based on the use of different silk fibroin solution inks such as the pure aqueous silk fibroin solution, 3D printing uses a silk fibroin solution incorporated with biologically active molecules and dopants, a self-curing silk fibroin solution, and a silk fibroin solution incorporated with living cells; and 3D printing in combination with sacrificial templating has been developed to fabricate 3D silk fibroin materials with complex structures down to the microscale (

B. mori silk fibroin materials applications
Attractive properties including light weight, excellent mechanical property, flexibility, optical transparency, thermal stability, biocompatibility, controllable biodegradability, and versatility of silk fibroin along with progress in biomedicine and nanofabrication technologies have expanded the scope of functionalized silk fibroin-based materials applications to medical, electronic, optical, and filtration applications.

Biomedical applications
In the biomedical field, current routine clinical uses of silk fibroin, working directly with the degummed silk fiber, include silk surgical mesh for abdominal wall reconstruction and investigative plastic surgery applications, silk fabrics for the treatment of dermatological conditions, and silk sutures for use in general soft tissue approximating ligation. Present clinical trials using regenerated silk fibroin materials include water-resistant silk films for wound healing and thin silk films for acute and chronic tympanic membrane perforation repair [5]. In addition to these routine clinical uses and clinical trials, regenerated silk fibroin-based materials in a wide spectrum of formats have been investigated as tissue engineering scaffolds and drug carriers in biomedical applications in preclinical studies. Until now, SF has been fabricated in a variety of materials such as nanofibrous mats, hydrogels, and 3D porous scaffolds for use in the engineering and regeneration of a variety of tissues including skin, nerves, tendons, blood vessels, bone, cartilage, ligaments, and tendon (Table 3).
Electrospun silk fibroin nonwoven mats consisting of nanofibers are remarkable tissue engineering materials since their extracellular matrix (ECM)-like fibrous nature is suitable for cell interaction (Figure 6a) [92]. And functional silk fibroin nanofibrous mats can be fabricated by the incorporation of growth factors or other signaling factors [62,96]. By controlling the gelation process (Figure 6b), blending with other biopolymers ( Figure  6c) and incorporating biologically functional moieties ( Figure  6d), silk fibroin hydrogels or sponges with fine-tuned stiffness and optimized cell functions can be constructed for specialized tissue engineering applications [93][94][95]. Besides mainly being http://www.autexrj.com/ used as injectable tissue fi llers, silk fi broin hydrogels with fi nely tuned properties can also be used for hard tissue regeneration [79,80,94]. Silk fi broin 3D biodegradable scaffolds taking the role of ECM analogues can serve as a necessary template or matrix supporting cell attachment, proliferation, differentiation, tissue neogenesis, formation of a new extracellular matrix, and transportation of nutrients and metabolic wastes [83-85, 94, 95, 98, 99]. It is worth noting that each type of engineered tissue should possess the correct balance of structural features and biological and mechanical properties for the induction of the desired cellular activity and to guide appropriate tissue regeneration.
Due to their combination of robust mechanical properties, controllable biodegradability and drug stabilization effect, silk fi broin-based materials have been investigated as sustainable drug delivery vehicles for a wide range of biological molecules including proteins, growth factors, peptides, and gene and therapeutic drugs such as anticancer agents and small molecule drugs [2,66,77]. Silk fi broin-based materials in the format of nanoparticles, electrospun nanofi brous mats, hydrogels, aerogels, coatings, and microneedles have been implemented as drug delivery platforms. Silk fi broin-based nanoparticles have been investigated as drug carriers due to their high binding capacity for various drugs, controlled drug release properties and mild preparation conditions.  By developing green synthesis methods and adjusting the particle size, the chemical structure, and properties, modified or recombinant nontoxic silk fibroin-based nanoparticles can be designed to improve the therapeutic efficiency of drugs encapsulated into these nanoparticles [100][101][102][103][104]. Silk fibroinbased nanofibrous mats have been used as potential carriers for drug delivery due to higher drug encapsulation efficiency and better stability than other drug formulations. By manipulating the crystallinity and biodegradation rate of silk fibroin via functionalization and optimizing the porosity, morphology, and diameter of nanofibers, silk fibroin-based nanofibrous mats with controlled drug release and enhanced cell behaviors can be obtained (Figure 7a) [105]. Moreover, high porosity and good interconnectivity between the pores of electrospun nanofibrous mats make them suitable for use as mass transporters [106]. Silk fibroin hydrogel, with a microenvironment similar to the ECM, is often used as a drug carrier due to its high affinity to these drugs and its molecular permeability. Research has been focused on fabrication of silk fibroin-based composite hydrogels with tuned properties, stimuli responsiveness, and optimized treatment effect. Niu et al. fabricated a newer type of drug releasing enzymatically crosslinked hydrogel based on a semi-interpenetrating network of silk fibroin and poly(vinyl alcohol) (PVA) (Figure 7b). The fabricated composite hydrogel was found to possess lamellar structure and release properties [107]. Akrami-Hasan-Kohal, et al. fabricated a sonicationassisted cross-linking silk fibroin hydrogel/dexamethasone sodium phosphate loaded chitosan nanoparticles with ideal physical properties and long-term drug release [108]. Silk fibroin-based films and porous scaffolds such as aerogels have also been used as drug carriers because of their open-pore network and structure tenability. The porosity and surface area of the device may affect its drug release kinetics [109][110][111]. Silk fibroin coatings have also been combined with liposomes for long-term and targeted drug delivery (Figure 7c) [112,113]. Due to its unique combination of favorable mechanical strength and ability to maintain the activity of encapsulated bioactive factors, the silk fibroin-based microneedles have been explored as a minimally invasive platform for delivering drugs or vaccines in a more patient-friendly manner. For example, Shin et al. fabricated oblique and sharp silk fibroin microneedles by digital light processing 3D printing for controllable transdermal drug delivery. The fabricated microneedles can realize effective transdermal delivery and controllable release of drug molecules [114]. Drug delivery devices made of silk fibroin may be found in a variety of material formats. Their choice depends on the mode of application, processability, desired release kinetics, and stability of the drug. Facile and green strategies should be developed to fabricate nontoxic silk fibroin-based materials with tunable properties and stimuli responsiveness for effective drug release.

Electronic applications
Flexible electronic devices are necessary for applications involving unconventional interfaces, such as soft and curved biological systems, where traditional silicon-based electronics present a mechanical mismatch [115]. By virtue of its light weight; excellent mechanical, dielectric, and optical properties; superior biosustainability and biodegradability; and ease of fabrication, silk fibroin has been utilized as the active element in the design of high-performance flexible electronics devices for a wide range of applications (Figure 8).
Silk fibroin has been combined with conductive materials such as carbon materials, ionic liquid, conducting polymers, and metal nanowires for sensor, electronic storage devices, electrodes, polymer electrolytes, and transient electronics applications, as listed in Table 4.
Although silk fibroin can be used as passive substrates and dielectric layers in electronic devices, the incorporated conductive components limited their biodegradability and bioresorbability for applications in biological systems [13,115].

Optical applications
In addition to applications in electronic devices, silk fi broin has also been heavily investigated as an interesting optical material because of its high transparency and excellent mechanical properties. Silk fi broin has been used in a variety of optical devices, such as optical diffraction-based sensors, optical waveguides, optical gazes, and 3D photonics. For example, Zhou et al. fabricated a set of bioactive optical diffraction-based sensors microfabricated using functionalized silk fi broin fi lms for optical diffraction-based sensing applications, including hydration sensing, biological concealment, therapeutic treatment, and in vitro and in vivo drug release monitoring upon degradation. Their work sheds light on a new class of transient optical devices -with specifi c functionalitiesthat can physically degrade in the body or disappear in the environment at prescribed times and at controlled rates ( Figure  9a) [122]. Li et al. fabricated silk fi broin-coated magnesium oxide nanospheres for noninvasive bioimaging applications (Figure 9b) [123]. The biocompatibility of waveguide material is important when it needs to interface directly with living cells in biomedical applications. Biocompatible silk fi broin-based optical waveguides have been produced by Santos et al. using direct laser writing and Prajzler using direct depositing ( Figure  9c), respectively [124,125]. Lee et al. fabricated silk fi broin fi lms and patterned them into different nanostructures via tipenhanced near-fi eld infrared nanolithography for rewritable optical storage medium [73].

Filtration systems
Among various material formats, materials prepared from electrospun nanofi bers have properties such as high surface-to-volume ratio, low pressure drop, and controllable morphology and connectivity, making them attractive for achieving excellent fi ltering performance. Because of their biocompatibility, biodegradability, and nontoxicity, silk fi broin materials in the form of electrospun nanofi brous mats have also been investigated as fi ltration systems for water treatment and air fi ltration. For example, Garrido et al. fabricated electrospun silk fi broin/ZnO mats to remove pesticide residues from water under natural sunlight [126]. Zhou et al fabricated a silk fi broingraphene oxide functionalized melamine sponge for effi cient oil absorption and oil and water separation [127]. Wang, et al. fabricated porous ZnS and ZnO fi lms using silk fi broin as a template by coaxial electrospinning for highly effi cient photodegradation of organic dyes (Figure 10a) [128]. Gao et al., [129] Min et al., [130] respectively fabricated electrospun silk nanofi bers for effi cient PM 2.5 and submicron particles capture (Figure 10b). Multifunctional silk fi broin nanofi berbased systems showed superior fi ltration performance with a much lower basis weight compared with commercial fi lters and absorbents. Yet, further efforts should be devoted to enhance the mechanical properties of the nanofi bers for longer life and design more advanced fi ltration system.

Conclusion and outlook
The ubiquity, unique hierarchical structure, light weight, excellent mechanical property, fl exibility, optical transparency, thermal stability, biocompatibility, controllable biodegradability, versatility in material format design, and mild aqueous processing of silk fi broin make silk fi broin-based materials an attractive biomaterial for various applications. Great efforts have been made to develop green and facile technologies for reverse-engineered silk fi broin extraction and materials fabrication. Silk fi broin-based materials in versatile formats, including fi bers, particles, fi lms, hydrogels, and 3D materials, have been developed for advanced utilities in medical, electronic, optical, and fi ltration applications. Bionanotechnology such as nanoimprinting/patterning, confi ned alignment, and 3D printing will further provide synergies for the prospective development of silk fi broin-based materials in related applications. Nevertheless, despite the immense level of development of silk fi broin-based materials, challenges in the large-scale production of silk fi broin and the control over the nanostructure of silk fi broin-based materials remain. Most of silk fi broin materials-processing techniques are still timeand solvent-intensive. Better control over the hierarchical structure to produce structurally and functionally optimized silk nanomaterials for specific applications is still a problem.