Aqueous Silk Fibroin Solution Research Articles

Silk‐Based Functional Inks with Color‐Tunable Light Upconversion for Printing on Arbitrary Surfaces

AbstractA biocompatible and versatile silk‐based functional ink with hydrophilic upconversion nanoparticles (UCNPs) that can be printed or painted on arbitrary substrates, including biological surfaces is demonstrated. Hydrophilic UCNPs, ready to be dispersed in aqueous silk fibroin solution, are synthesized in large quantities via facile hydrothermal synthesis using citric acid. Based on a systematic study of dopant control to achieve robust red emission from citrate‐capped UCNPs, three primary colors are obtained, which serve as the basis for the expansion of the color palette. The synthesized UCNPs are homogeneously dispersed in regenerated silk fibroin solutions derived from Bombyx mori cocoons to produce water‐based functional inks. Cyan, magenta, and yellow (CMY) inks are created by combining red, green, and blue (RGB) inks. Demonstrator devices, where a variety of colorful patterns and codes are revealed only under near‐infrared irradiation, are realized through stencil printing or brush painting on flexible polymer films, fruit, leaves, silkworm cocoons, and pig skin. The patterns and codes are highly heat resistant (≈230 °C) and can be easily washed off with water after use. The printable/paintable inks and functions proposed here will provide new opportunities for applications where sustainability, biocompatibility, and intentional luminescence are simultaneously required.

Ice Recrystallization Inhibition Activity of Silk Proteins.

The cryopreservation of cells, tissue, and organs is essential in both fundamental research and practical applications, such as modern regenerative medicine and technological applications. However, the formation of ice crystals during ice recrystallization can have harmful or even fatal effects on biological systems. To address this challenge, we explore the ice recrystallization inhibition (IRI) activity of two natural silk proteins of Bombyx mori, fibroin and sericin. We found that silk fibroin (SF) had higher ice recrystallization inhibition activity than silk sericin (SS). Moreover, SF aqueous solutions perform better in inhibiting ice recrystallization than SF phosphate-buffered saline solutions. Sum-frequency generation spectroscopy shows that stronger electrostatic interactions are responsible for the higher IRI ability of SF. This work is significant for broadening the applications of silk proteins in biomedical fields.

Triptorelin nanoparticle-loaded microneedles for use in assisted reproductive technology

Triptorelin is a first-line drug for assisted reproductive technology (ART), but the low bioavailability and frequent subcutaneous injection of triptorelin impair the quality of life of women preparing to become pregnant. We report silk fibroin (SF)-based microneedles (MNs) for transdermal delivery of triptorelin-loaded nanoparticles (NPs) to improve bioavailability and achieve safe and efficacious self-administration of triptorelin. Triptorelin was mixed into an aqueous solution of SF with shear force to prepare NPs to control the release and avoid the degradation of triptorelin by enzymes in the skin. Two-step pouring and centrifugation were employed to prepare nanoparticles-encapsulated polymeric microneedles (NPs-MNs). An increased β-sheet content in the conformation ensured that NPs-MNs had good mechanical properties to pierce the stratum corneum. Transdermal release of triptorelin from NPs-MNs was increased to ∼65%. The NPs-MNs exhibited a prolonged drug half-life and increased relative bioavailability after administration to rats. Surging levels of luteinizing hormone and estradiol in plasma and their subsequent prolonged downregulation indicate the potential therapeutic role of NPs-MNs in ART regimens. The triptorelin-loaded NPs-MNs developed in this study may reduce the physical and psychological burden of pregnant women using ART regimens.

Mechanically Robust Shape‐Memory Organohydrogels Based on Silk Fibroin with Organogel Microinclusions of Various Sizes

AbstractOrganohydrogels (OHGs) are soft materials with antagonistic hydrophilic and hydrophobic domains that have great interest for many different applications. This study presents the preparation of mechanically strong OHGs with shape‐memory function by incorporating semicrystalline organo‐microgels within the pores of silk fibroin (SF) scaffolds. In the first step, SF cryogels with various pore diameters between 26 ± 8 and 17 ± 4 µm are synthesized by cryogelation of aqueous SF solutions at concentrations between 5 and 20 w/v%. In the second step, the pores of SF scaffolds are filled with an organogel precursor solution containing n‐octadecyl acrylate (C18A), acrylic acid, N,N'‐methylene(bis)acrylamide, and an initiator. Once the free‐radical polymerization took place inside the pores, OHGs containing organo‐microgels of various sizes are obtained. The incorporation of the organogel component in the cryogels generates crystalline areas due to the side‐by‐side packed C18 side chains. OHGs' melting temperature and crystallinity level can be varied from 42 to 54 °C and from 2 to 16%, respectively. The stiffness of OHGs increases from 5.9 ± 0.5 to 18 ± 1 MPa with increasing SF concentration from 5 to 20 w/v%, which is attributed to the decreasing pore size of the cryogels and increasing thickness of the pore walls.

Unidirectional Nanopore Dehydration Induces a Highly Stretchable and Mechanically Robust Silk Fibroin Membrane Dominated by Type II β-Turns.

Aqueous silk fibroin solution is dehydrated by evaporation into a water-soluble cast film (SFME) with poor mechanical properties but becomes by unidirectional nanopore dehydration (UND) into silk fibroin membrane (SFMU) with water-stable and good mechanical robustness. The thickness and tensile force of the SFMU are almost twice those of the MeOH-annealed SFME. The UND-based SFMU has a tensile strength of 15.82 MPa, an elongation of 665.23%, and a type II β-turn (Silk I) that accounts for 30.75% of the crystal structure. Mouse L-929 cells adhere, grow, and proliferate well on it. The UND temperature can be used to tune the secondary structure, mechanical properties, and biodegradability. UND induced the oriented arrangement of the silk molecules, which led to the formation of the SFMU dominated with Silk I structure. The silk metamaterial by controllable UND technology has great potential in medical biomaterials, biomimetic materials, sustained drug release, and flexible electronic substrates.

Silk Fibroin-Based Shape-Memory Organohydrogels with Semicrystalline Microinclusions.

Inspired by nature, we designed organohydrogels (OHGs) consisting of a silk fibroin (SF) hydrogel as the continuous phase and the hydrophobic microinclusions based on semicrystalline poly(n-octadecyl acrylate) (PC18A) as the dispersed phase. SF acts as a self-emulsifier to obtain oil-in-water emulsions, and hence, it is a versatile and green alternative to chemical emulsifiers. We first prepared a stable oil-in-water emulsion without an external emulsifier by dispersing the n-octadecyl acrylate (C18A) monomer in an aqueous SF solution. To stabilize the emulsions for longer times, gelation in the continuous SF phase was induced by the addition of ethanol, which is known to trigger the conformational transition in SF from random coil to β-sheet structures. In the second step, in situ polymerization of C18A droplets in the emulsion system was conducted under UV light in the presence of a photoinitiator to obtain high-strength OHGs with shape-memory function, and good cytocompatibility. The incorporation of hydrophilic N,N-dimethylacrylamide and noncrystallizable hydrophobic lauryl methacrylate units in the hydrogel and organogel phases of OHGs, respectively, further improved their mechanical and shape-memory properties. The shape-memory OHGs presented here exhibit switchable viscoelasticity and mechanics, a high Young's modulus (up to 4.3 ± 0.1 MPa), compressive strength (up to 2.5 ± 0.1 MPa), and toughness (up to 0.68 MPa).

Bubble-blowing-inspired sub-micron thick freestanding silk films for programmable electronics.

Thin film electronics that are capable of deforming and interfacing with nonplanar surfaces have attracted widespread interest in wearable motion detection or physiological signal recording due to their light weight, low stiffness, and high conformality. However, it is still a challenge to fabricate freestanding thin film substrates or matrices with only sub-micron thickness in a simple way, especially for those materials with metastable conformations, like regenerated silk protein. Herein, we developed a dip-coating method for the fabrication of sub-micron thick freestanding silk films inspired by blowing soap bubbles. Using a closed-loop frame to dip-coat in a concentrated silk fibroin aqueous solution, the substrate-free silk films with a thickness as low as hundreds of nanometres (∼150 nm) can be easily obtained after solvent evaporation. The silk films have extremely smooth surfaces (Rq < 3 nm) and can be tailored with different geometric shapes. The naturally dried silk films possess random coil dominated uncrystallized secondary structures, exhibiting high modulation ability and adaptability, which can be conformally attached on wrinkled skin or wrapped on human hair. Considering the methodological advantages and the unique properties of the obtained sub-micron thick silk films, several thin film based programmable electronics including transient/durable circuits, skin electrodes, transferred skin light-emitting devices and injectable electronics are successfully demonstrated after being deposited with gold or conducting polymer layers. This research provides a new avenue for preparing freestanding thin polymer films, showing great promise for developing thin film electronics in wearable and biomedical applications.

Synthesis of hollow fibroin using calcium carbonate as a template

AbstractSilk fibroin is a unique biomaterial and has gained much attention in biomedical applications. In this study, we have demonstrated the formation of hollow fibroin using the selective etching method. Initially, spherical CaCO3 was synthesized in the presence of polyelectrolyte, poly(4‐styrenesulfonate). CaCO3 particles were treated with aqueous silk fibroin solution, followed by methanol treatment. The sequential deposition of silk fibroin was achieved through a layer‐by‐layer (LbL) strategy to obtain fibroin‐encapsulated CaCO3. Finally, the fibroin‐ encapsulated CaCO3 was treated with an acid solution to form hollow fibroin. The crystalline phase of as‐prepared products was measured using powder X‐ray diffraction, and vibrational modes of products were characterized using Fourier transform infrared spectroscopy. The hollow fibroin morphology was examined using electron microscopy. The resultant hollow fibroin has good stability and can be explored in various applications.

Silk fibroin/carbon nanofiber composite aerogel for efficient and stable solar steam generation

The development of bio-based aerogel is an important solar evaporation issue owing to its environmental protection, sustainable water transportation, and intrinsic thermal management properties. Herein, silk fibroin (SF)/carbon nanofiber (CN) composite aerogel (SCNA) was prepared by incorporating carbonized electrospun polyacrylonitrile (PAN) nanofibers into SF aqueous solution and then performing a freeze-drying process. The SCNA with optimized CN loading demonstrated outstanding solar absorbing capacity (99.89%) across the whole spectrum due to the synergy between the functional solar absorbing CN material and the SF skeleton (71.64%). With high porosity, the SF skeleton can encapsulate the CN, transport water, and suppress heat loss in solar evaporation. As a result, high evaporation rates were achieved for pure water (1.46 kg m−2 h−1), 3.5% salty water (1.42 kg m−2 h−1), and actual dyeing wastewater (1.43 kg m−2 h−1). Besides, the SCNA could be reused more than ten times without performance degradation, assuring long-term efficient solar steam generation. Therefore, this work will stimulate more research on the use of biomass materials for solar evaporation, especially silk-based aerogel.

Antitumor Effect and Pharmacological Mechanism of Paclitaxel-loaded Silk Fibroin Nanomaterials on H22 Subcutaneous Tumor Model of Mouse Hepatoma

This work was developed to analyze the adoption of paclitaxel-loaded silk fibroin nanoparticles (SFNPs) in subcutaneous transplanted tumor model of mouse hepatoma to explore its anti-tumor effect. Twenty-five specific pathogen-free (SPF) mice were selected to construct a subcutaneous tumor model of liver cancer, and they were randomly rolled into control group, group A, group B, group C, and group D, with five mice in each group. The silk fibroin was mixed with an organic solvent to prepare a suspension, and the SFNPs were prepared through centrifugation, ultrasound, and other methods. The paclitaxel-loaded SFNPs were prepared by mixing paclitaxel and silk fibroin aqueous solution, centrifuging, washing, and dispersing. Then, the five groups of mice were intervened by different dosage regimens to analyze the changes of various indicators. As a result, the prepared nanoparticles had uniform particle size, uniform distribution, no adhesion, and the average particle size was less than 500 nm. The tumor volume of mice in groups C and D on the 7th, 9th, and 13th days of administration were dramatically smaller than those in the control group, group A, and group B (P [ 0.05). And the tumor volume (154.49 � 9.65 mm3) of mice in group D on the 13th day of administration was dramatically smaller than that in group C (167.79 � 9.72 mm3) (P [ 0.05). The tumor mass (0.89 � 0.14 g, 0.54 � 0.13 g, and 0.46 � 0.11 g) of mice in groups B, C, and D was dramatically smaller than that in the control group and group A (1.23 � 0.12 g, 1.24 � 0.11 g) (P [ 0.05), and that of group D was dramatically smaller than groups B and C (P [ 0.05). The apoptosis rates of tumor cells in groups C and D (46.38%, 48.23%) were greatly superior to those in the control group (16.7%), group A (21.33%), and group B (35.6%) (P [ 0.05), and that of group D was greatly superior to that in group C (P [ 0.05). In summary, paclitaxel-loaded SFNPs can effectively improve the targeting effect and bioavailability of drugs in the treatment of liver cancer, thereby improving the efficacy, and had a good application prospect.

Amino Acid Surfactant‐Induced Superfast Gelation of Silk Fibroin for Treating Noncompressible Hemorrhage

AbstractThe gelation of silk fibroin (SF) aqueous solution cross‐linked by externally induced β‐sheets generally takes longer times, and requires relatively strict conditions, limiting their biomedical application possibilities. Here, a simplified and straightforward strategy is proposed to induce superfast gelation of SF by a biocompatible Food and Drug Administration‐approved amino acid surfactant, ethyl lauroyl arginine hydrochloride (LAE). The gelation time is as short as 15 s at 60 °C and falls within 1 min around body temperature. The distinct pathway and thermodynamics of superfast gelation of SF is deciphered, and three important factors including superfast and complete unfolding, heterogeneous nucleation seeds, and faster formation of more β‐sheets that are crucial for superfast SF assembly are revealed. The LAE‐induced in situ superfast gelation mechanism is first harnessed to fabricate an injectable antibacterial biodegradable hemostatic hydrogel for treatment of noncompressible liver bleeding. The amphiphilicity of LAE and superfast gelation is exploited to quickly entrap different sizes of air bubbles in the upper and lower part of the hydrogel, which is then lyophilized to form an asymmetric hierarchical porous SF‐LAE sponge, where the large pores serve to drain blood, and dense pores prevent the outflow of blood to seal the wound.

Silk fibroin microfiber‐reinforced polycaprolactone composites with enhanced biodegradation and biological characteristics

There is an enormous demand for bone graft biomaterials to treat developmental and acquired bony defects arising from infections, trauma, tumor, and other conditions. Polycaprolactone (PCL) has been extensively utilized for bone tissue engineering but limited cellular interaction and tissue integration are the primary concerns. PCL-based composites with different biomaterials have been attempted to improve the mechanical and biological response. Interestingly, a few studies have tried to blend PCL with aqueous silk fibroin solution, but the structures prepared with the blend were mechanically weak due to phase mismatch. As a result, silk microparticle-based PCL composites have been prepared, but the microfibers-reinforced composites could be superior to them due to significant fiber-matrix interaction. This study aims at developing a unique composite by incorporating 100-150μm long (aspect ratio; 8:1-5:1) silk-fibroin microfibers into the PCL matrix for superior biological and mechanical properties. Two silk variants were used, that is, Bombyx mori and a wild variant, Antheraea mylitta, reported to have cell recognizable Arginine-Glycine-Aspartic acid (RGD) sequences. A.mylitta silk fibroin microfibers were produced, and composites were made with PCL for the first time. The morphological, tensile, thermal, biodegradation, and biological properties of the composites were evaluated. Importantly, we tried to optimize the silk concentration within the composite to strike a balance among the cellular response, biodegradation, and mechanical strength of the composites. The results indicate that the PCL-silk fibroin microfiber composite could be an efficient biomaterial for bone tissue engineering.

Gelation mechanism of regenerated silk fibroin aqueous solution during storage

Gelation mechanism of regenerated silk fibroin aqueous solution during storage

Effect of pH on the Conformational Transition of Silk Fibroin in Aqueous Solution Monitored by Thioflavin-T Fluorescence.

In this work, the potential application of the fluorescence dye Thioflavin-T (ThT), which can specifically bind to amyloid, as a powerful tool for monitoring secondary structural transitions of silk fibroin (SF) induced by pH in low solution concentrations was examined. Results showed that ThT emission intensities substantially increased when pH decreased from 6.8 to 4.8. This increase may be ascribed to conformational transitions from random coil to β-sheet. The morphology and secondary structure of SF were also investigated via TEM, AFM and circular dichroism spectroscopy. The information obtained herein can be utilized not only for the development of convenient and efficient noninvasive method for monitoring the assembly behavior of SF in aqueous solution but also for in vitro fluorescence imaging.

Fractionation of Regenerated Silk Fibroin and Characterization of the Fractions.

The molecular weight (MW) of regenerated silk fibroin (RSF) decreases during degumming and dissolving processes. Although MW and the MW distribution generally affect polymer material processability and properties, few reports have described studies examining the influences of MW and the distribution on silk fibroin (SF) material. To prepare different MW SF fractions, the appropriate conditions for fractionation of RSF by ammonium sulfate (AS) precipitation process were investigated. The MW and the distribution of each fraction were found using gel permeation chromatography (GPC) and SDS-polyacrylamide electrophoresis (SDS-PAGE). After films of the fractionated SFs formed, the secondary structure, surface properties, and cell proliferation of films were evaluated. Nanofiber nonwoven mats and 3D porous sponges were fabricated using the fractionated SF aqueous solution. Then, their structures and mechanical properties were analyzed. The results showed AS precipitation using a dialysis membrane at low temperature to be a suitable fractionation method for RSF. Moreover, MW affects the nanofiber and sponge morphology and mechanical properties, although no influence of MW was observed on the secondary structure or crystallinity of the fabricated materials.

A Fast and Reliable Process to Fabricate Regenerated Silk Fibroin Solution from Degummed Silk in 4 Hours.

Silk fibroin has a high potential for use in several approaches for technological and biomedical applications. However, industrial production has been difficult to date due to the lengthy manufacturing process. Thus, this work investigates a novel procedure for the isolation of non-degraded regenerated silk fibroin that significantly reduces the processing time from 52 h for the standard methods to only 4 h. The replacement of the standard degumming protocol by repeated short-term microwave treatments enabled the generation of non-degraded degummed silk fibroin. Subsequently, a ZnCl2 solution was used to completely solubilize the degummed fibroin at only 45 °C with an incubation time of only 1 h. Desalting was performed by gel filtration. Based on these modifications, it was possible to generate a cytocompatible aqueous silk fibroin solution from degummed silk within only 4 h, thus shortening the total process time by 48 h without degrading the quality of the isolated silk fibroin solution.

A Comparative Analysis of the Structure and Biological Properties of Films and Microfibrous Scaffolds Based on Silk Fibroin.

A comparative analysis of the structure and biological properties of silk fibroin constructions was performed. Three groups of constructions were obtained: films obtained by casting an aqueous solution of silk fibroin and electrospun microfibrous scaffolds based on silk fibroin, with the addition of 30% gelatin per total protein weight. The internal structures of the films and single fibers of the microfibrous scaffolds consisted of densely packed globule structures; the surface area to volume ratios and volume porosities of the microfibrous scaffolds were calculated. All constructions were non-toxic for cells and provide high levels of adhesion and proliferation. The high regenerative potential of the constructions was demonstrated in a rat full-thickness skin wound healing model. The constructions accelerated healing by an average of 15 days and can be considered to be promising constructions for various tasks of tissue engineering and regenerative medicine.

Low voltage electric field governs fibrous silk electrogels

Silk fibroin gels, formed with electric fields, have been studied and potential utility in medical materials. However, silk electrogels with controllable nanofiber network morphology have rarely been reported. In this work, low voltage electric field was developed to control the nanofiber network morphology formation and resulting electrogelation of silk fibroin aqueous solution, inducing a transition from an overall protein structure that is initially rich in random coil to one that is rich in β-sheet. Under low voltage (3–24 V), solution concentration (≤5.0 wt%), and neutral, alkaline conditions, silk fibroin fibrous electrogels were easily formed. At the same time, compression test result exhibited the compressive stress of silk fibroin fibrous electrogels was 250.17 ± 16.50 kPa at dry state, which formed from silk fibroin concentration 3.0 wt%. Moreover, the recovery rate of electrogels can reach more than 50% after 10 compression-recovery circles, exhibiting excellent flexibility. In addition, silk fibroin electrogels loaded cefixime drugs have obvious antibacterial effect against Staphylococcus aureus and Escherichia coli than commercial medical band-aids. Drug-loaded silk fibroin fibrous electrogels supported mouse embryonic fibroblasts 3 T3 cells attachment and proliferation over 7 days. This study offers an effective approach to design and synthesis of new dressings.

Formation, Structure, and Mechanical Performance of Silk Nanofibrils Produced by Heat-Induced Self-Assembly.

The heat-induced self-assembly of silk fibroin (SF) is studied by combing fluorescence assessment, infrared nanospectroscopy, wide-angle X-ray scattering, and Derjaguin-Muller-Toporov coupled with atomic force microscopy. Several fundamental issues regarding the formation, structure, and mechanical performance of silk nanofibrils (SNFs) under heat-induced self-assembly are discussed. Accordingly, SF in aqueous solution is rod-like in shape and not micellar. The formation of SNFs occurs through nucleation-dependent aggregation, but the assembly period is variable and irregular. SF shows inherent fractal growth, and this trend is critical for the short-term assembly. The long-term assembly of SF, however, mainly involves an elongation growth process. SNFs produced by different methods, such as ethanol treatment and heat incubation, have similar secondary structure and mechanical properties. These investigations improve the in-depth understanding of fundamental issues related to self-assembly of SNFs, and thus provide inspiration and guidance in designing of silk nanomaterials.

Stretchable silk fibroin hydrogels

Hydrogels derived from silk fibroin (SF) are attractive soft materials in biomedical applications such as drug delivery and tissue engineering. However, SF hydrogels reported so far are generally brittle in tension limiting their load-bearing applications. We present here a novel strategy for preparing stretchable SF hydrogels by incorporating flexible polymer chains into the brittle SF network, which strengthen the interconnections between SF globules. We included N, N-dimethylacrylamide (DMAA) monomer and ammonium persulfate initiator into an aqueous SF solution containing a diepoxide cross-linker to in situ generate flexible poly (N,N-dimethylacrylamide) (PDMAA) chains. Moreover, instead of SF, methacrylated SF was used for the gel preparation to create an interconnected SF/PDMAA network. The free-radical polymerization of DMAA leads to the formation of PDMAA chains interconnecting globular SF molecules via their pendant vinyl groups. Incorporation of 2 w/v% DMAA into the SF network turns the brittle hydrogel into a stretchable one sustaining up to 370% elongation ratio. The mechanical properties of SF hydrogels could be adjusted by the amount of PDMAA incorporated into the SF network. The stretchable and tough SF hydrogels thus developed are suitable as a scaffold in tissue engineering and offer an advantage as a biomaterial over other SF-based biomaterials.