Metal ions guide the production of silkworm silk fibers.

Silk fibers' unique mechanical properties have made them desirable materials, yet their formation mechanism remains poorly understood. While ions are known to support silk fiber production, their exact role has thus far eluded discovery. Here, we use cryo-electron microscopy coupled with elemental analysis to elucidate the changes in the composition and spatial localization of metal ions during silk evolution inside the silk gland. During the initial protein secretion and storage stages, ions are homogeneously dispersed in the silk gland. Once the fibers are spun, the ions delocalize from the fibroin core to the sericin-coating layer, a process accompanied by protein chain alignment and increased feedstock viscosity. This change makes the protein more shear-sensitive and initiates the liquid-to-solid transition. Selective metal ion doping modifies silk fibers' mechanical performance. These findings enhance our understanding of the silk fiber formation mechanism, laying the foundations for developing new concepts in biomaterial design.

Immunomodulatory Antibacterial Hydrogel for Wound Infection Management.

Background: Wound healing has always been a focal point in clinical work. Bacterial infections and immune microenvironment disorders can both hinder normal wound healing. Current wound dressings only serve a covering function. Developing wound dressings with antibacterial and immunomodulatory functions is crucial for aiding wound healing. To address this issue, we have developed a hydrogel with antibacterial and immunomodulatory functions for managing infected wounds. Methods: The present study describes a photo-crosslinked antibacterial hydrogel composed of curcumin, silver nanoparticles-loaded reduced graphene oxide, and silk fibroin methacryloyl for the treatment of infected wounds. The study assessed its antibacterial properties and its capacity to induce macrophage M2 polarization through in vitro and in vivo experiments. Results: The hydrogel demonstrates robust antibacterial properties and enhances macrophage M2 polarization in both in vitro and in vivo settings. Moreover, it accelerates the healing of infected wounds in vivo by stimulating collagen deposition and angiogenesis. Conclusion: Overall, this hydrogel shows great potential in managing wound infections.

Formulations of poly(vinyl alcohol) functionalized silk fibroin nanoparticles for the oral delivery of zwitterionic ciprofloxacin.

Fibroin nanoparticles (FNP) have been employed in numerous biomedical applications. However, limited research has focused on the oral delivery of FNP and in-depth molecular interactions between the encapsulated drug and FNP. Therefore, this work developed the FNP, functionalized with poly(vinyl alcohol) (PVA), to orally deliver the zwitterionic ciprofloxacin, focused on the molecular interactions. The particles were formulated using both desolvation (the drug precipitated during the particles formulation) and adsorption (the drug adsorbed on the particles surfaces) methods. The optimal formula possessed a size of ~630 nm with narrow size distribution (measured by DLS method), spherical shape (determined by SEM), and moderate drug loading (confirmed by FT-IR, XRD, and DSC techniques) of ~50% for the desolvation method and ~43% for the adsorption method. More than 80% of the drug molecules resided on the particle surfaces, mainly via electrostatic forces with fibroin. The drug was physically adsorbed onto FNP, which followed Langmuir model and pseudo second-order kinetics. In the in-vitro simulated gastric condition at pH 1.2, the ciprofloxacin bound strongly with FNP via electrostatic forces, thus hindering the drug release (< 40%). Contrastingly, in the simulated intestinal condition at pH 6.8, the particles could control the drug release rates dependent on the PVA amount, with up to ~100% drug release. Lastly, the particles possessed adequate antibacterial activities on Bacillus subtilis, Escherichia coli, and Salmonella enterica, with MIC of 128, 8, and 32 µg/mL, respectively. In summary, the FNP and PVA functionalized FNP could be a potential oral delivery system for zwitterionic drugs.

Silk Fibroin Microneedles Loaded with Lipopolysaccharide-Pretreated Bone Marrow Mesenchymal Stem Cell-Derived Exosomes for Oral Ulcer Treatment.

Oral ulcers, superficial lesions on the surface of the oral mucosa, have a high incidence rate, and their main symptoms include local pain and erosion. Lipopolysaccharide (LPS)-preconditioned bone marrow mesenchymal stem cells and their secreted exosomes (LPS-pre-Exos) have been shown to promote recovery in various inflammatory conditions and wounds. However, studies documenting LPS-pre-Exos as a therapeutic intervention for oral mucosal-like diseases are lacking. In this study, we prepared a silk fibroin microneedle (MN) patch consisting of LPS-pre-Exos and zeolitic imidazolate framework-8 (ZIF-8) that localized at the tip and base, respectively, and used this MN patch for oral ulcer treatment. Upon insertion into the oral mucosa, continuous LPS-pre-Exos release was observed, which promoted macrophage polarization and tissue healing. Additionally, the ZIF-8 framework in the MN patch facilitated the controlled release of Zn2+, which demonstrated potent antimicrobial properties via synergistic effects. The in vitro experimental results showed that the silk fibroin MN patch can continuously release LPS-pre-Exos and Zn2+ for more than 7 days. Thus, the LPS-pre-Exos and ZIF-8-loaded silk fibroin MN patch exhibited good anti-inflammatory and antibacterial properties, promoting oral ulcer healing, and showed good histocompatibility. Hence, it may represent a potentially valuable strategy for facilitating oral ulcer healing.

Electrical stimulation enhances sciatic nerve regeneration using a silk-based conductive scaffold beyond traditional nerve guide conduits.

Despite recent advancements in peripheral nerve regeneration, the creation of nerve conduits with chemical and physical cues to enhance glial cell function and support axonal growth remains challenging. This study aimed to assess the impact of electrical stimulation (ES) using a conductive nerve conduit on sciatic nerve regeneration in a rat model with transection injury. The study involved the fabrication of conductive nerve conduits using silk fibroin and Au nanoparticles (AuNPs). Collagen hydrogel loaded with green fluorescent protein (GFP)-positive adipose-derived mesenchymal stem cells (ADSCs) served as the filling for the conduit. Both conductive and non-conductive conduits were applied with and without ES in rat models. Locomotor recovery was assessed using walking track analysis. Histological evaluations were performed using H&E, luxol fast blue staining and immunohistochemistry. Moreover, TEM analysis was conducted to distinguish various ultrastructural aspects of sciatic tissue. In the ES + conductive conduit group, higher S100 (p < 0.0001) and neurofilament (p < 0.001) expression was seen after 6 weeks. Ultrastructural evaluations showed that conductive scaffolds with ES minimized Wallerian degeneration. Furthermore, the conductive conduit with ES group demonstrated significantly increased myelin sheet thickness and decreased G. ratio compared to the autograft. Immunofluorescent images confirmed the presence of GFP-positive ADSCs by the 6th week. Locomotor recovery assessments revealed improved function in the conductive conduit with ES group compared to the control group and groups without ES. These results show that a Silk/AuNPs conduit filled with ADSC-seeded collagen hydrogel can function as a nerve conduit, aiding in the restoration of substantial gaps in the sciatic nerve with ES. Histological and locomotor evaluations indicated that ES had a greater impact on functional recovery compared to using a conductive conduit alone, although the use of conductive conduits did enhance the effects of ES.

Multilevel neurium-mimetic individualized graft via additive manufacturing for efficient tissue repair.

Complicated peripheral nerve injuries or defects, especially at branching sites, remain a prominent clinical challenge after the application of different treatment strategies. Current nerve grafts fail to match the expected shape and size for delicate and precise branched nerve repair on a case-by-case basis, and there is a lack of geometrical and microscale regenerative navigation. In this study, we develop a sugar painting-inspired individualized multilevel epi-/peri-/endoneurium-mimetic device (SpinMed) to customize natural cues, featuring a selectively protective outer sheath and an instructive core, to support rapid vascular reconstruction and consequent efficient neurite extension along the defect area. The biomimetic perineurium dictates host-guest crosslinking in which new vessels secrete multimerin 1 binding to the fibroin filler surface as an anchor, contributing to the biological endoneurium that promotes Schwann cell homing and remyelination. SpinMed implantation into rat sciatic nerve defects yields a satisfactory outcome in terms of structural reconstruction, with sensory and locomotive function restoration. We further customize SpinMed grafts based on anatomy and digital imaging, achieving rapid repair of the nerve trunk and branches superior to that achieved by autografts and decellularized grafts in a specific beagle nerve defect model, with reliable biosafety. Overall, this intelligent art-inspired biomimetic design offers a facile way to customize sophisticated high-performance nerve grafts and holds great potential for application in translational regenerative medicine.

Enhanced effects of slowly co-released TGF-ß3 and BMP-2 from biomimetic calcium phosphate-coated silk fibroin scaffolds in the repair of osteochondral defects.

Bioactive agents have demonstrated regenerative potential for cell-free bone tissue engineering. Nevertheless, certain challenges persist, including ineffective delivery methods and confined therapeutic potency. Here, we demonstrated that the biomimetic calcium phosphate coating system (BioCaP) could effectively uptake and slowly release the incorporated bioactive agents compared to the surface absorption system via osteoclast-mediated degradation of BioCaP coatings. The release kinetics were determined as a function of time. The release rate was stable without remarkable burst release during the first 1 day, followed by a sustained release from day 7 to day 19. Then, we developed the bi-functional BioCaP-coated silk fibroin scaffolds enabling the effective co-delivery of TGF-ß3 and BMP-2 (SFI-T/SFI-B) and the corresponding slow release of TGF-ß3 and BMP-2 exhibited superior potential in promoting chondrogenesis and osteogenesis without impairing cell vitality in vitro. The SFI-T/SFI-B scaffolds could improve cartilage and bone regeneration in 5 × 4 mm rabbit osteochondral (OC) defect. These findings indicate that the biomimetic calcium-phosphate coated silk fibroin scaffolds with slowly co-released TGF-ß3 and BMP-2 effectively promote the repair of OC defects, hence facilitating the future clinical translation of controlled drug delivery in tissue engineering.

Molecular organization of fibroin heavy chain and mechanism of fibre formation in Bombyx mori.

Fibroins' transition from liquid to solid is fundamental to spinning and underpins the impressive native properties of silk. Herein, we establish a fibroin heavy chain fold for the Silk-I polymorph, which could be relevant for other similar proteins, and explains mechanistically the liquid-to-solid transition of this silk, driven by pH reduction and flow stress. Combining spectroscopy and modelling we propose that the liquid Silk-I fibroin heavy chain (FibH) from the silkworm, Bombyx mori, adopts a newly reported ß-solenoid structure. Similarly, using rheology we propose that FibH N-terminal domain (NTD) templates reversible higher-order oligomerization driven by pH reduction. Our integrated approach bridges the gap in understanding FibH structure and provides insight into the spatial and temporal hierarchical self-assembly across length scales. Our findings elucidate the complex rheological behaviour of Silk-I, solutions and gels, and the observed liquid crystalline textures within the silk gland. We also find that the NTD undergoes hydrolysis during standard regeneration, explaining key differences between native and regenerated silk feedstocks. In general, in this study we emphasize the unique characteristics of native and native-like silks, offering a fresh perspective on our fundamental understanding of silk-fibre production and applications.

De Novo Long-Read Genome Assembly and Annotation of the Luna Moth (Actias luna) Fully Resolves Repeat-Rich Silk Genes.

We present the first long-read de novo assembly and annotation of the luna moth (Actias luna) and provide the full characterization of heavy chain fibroin (h-fibroin), a long and highly repetitive gene (>20 kb) essential in silk fiber production. There are >160,000 described species of moths and butterflies (Lepidoptera), but only within the last 5 years have we begun to recover high-quality annotated whole genomes across the order that capture h-fibroin. Using PacBio HiFi reads, we produce the first high-quality long-read reference genome for this species. The assembled genome has a length of 532 Mb, a contig N50 of 16.8 Mb, an L50 of 14 contigs, and 99.4% completeness (BUSCO). Our annotation using Bombyx mori protein and A. luna RNAseq evidence captured a total of 20,866 genes at 98.9% completeness with 10,267 functionally annotated proteins and a full-length h-fibroin annotation of 2,679 amino acid residues.

Controlling Drug Partitioning in Individual Protein Condensates through Laser-Induced Microscale Phase Transitions.

Gelation of protein condensates formed by liquid-liquid phase separation occurs in a wide range of biological contexts, from the assembly of biomaterials to the formation of fibrillar aggregates, and is therefore of interest for biomedical applications. Soluble-to-gel (sol-gel) transitions are controlled through macroscopic processes such as changes in temperature or buffer composition, resulting in bulk conversion of liquid droplets into microgels within minutes to hours. Using microscopy and mass spectrometry, we show that condensates of an engineered mini-spidroin (NT2repCTYF) undergo a spontaneous sol-gel transition resulting in the loss of exchange of proteins between the soluble and the condensed phase. This feature enables us to specifically trap a silk-domain-tagged target protein in the spidroin microgels. Surprisingly, laser pulses trigger near-instant gelation. By loading the condensates with fluorescent dyes or drugs, we can control the wavelength at which gelation is triggered. Fluorescence microscopy reveals that laser-induced gelation significantly further increases the partitioning of the fluorescent molecules into the condensates. In summary, our findings demonstrate direct control of phase transitions in individual condensates, opening new avenues for functional and structural characterization.

NGF-BMSC-SF/CS composites for repairing knee joint osteochondral defects in rabbits: evaluation of the repair effect and potential underlying mechanisms.

BACKGROUND: With the rapid growth of the ageing population, chronic diseases such as osteoarthritis have become one of the major diseases affecting the quality of life of elderly people. The main pathological manifestation of osteoarthritis is articular cartilage damage. Alleviating and repairing damaged cartilage has always been a challenge. The application of cartilage tissue engineering methods has shown promise for articular cartilage repair. Many studies have used cartilage tissue engineering methods to repair damaged cartilage and obtained good results, but these methods still cannot be used clinically. Therefore, this study aimed to investigate the effect of incorporating nerve growth factor (NGF) into a silk fibroin (SF)/chitosan (CS) scaffold containing bone marrow-derived mesenchymal stem cells (BMSCs) on the repair of articular cartilage defects in the knees of rabbits and to explore the possible underlying mechanism involved. MATERIALS AND METHODS: Nerve growth factor-loaded sustained-release microspheres were prepared by a double emulsion solvent evaporation method. SF/CS scaffolds were prepared by vacuum drying and chemical crosslinking. BMSCs were isolated and cultured by density gradient centrifugation and adherent culture. NGF-SF/CS-BMSC composites were prepared and implanted into articular cartilage defects in the knees of rabbits. The repair of articular cartilage was assessed by gross observation, imaging and histological staining at different time points after surgery. The repair effect was evaluated by the International Cartilage Repair Society (ICRS) score and a modified Wakitani score. In vitro experiments were also performed to observe the effect of different concentrations of NGF on the proliferation and directional differentiation of BMSCs on the SF/CS scaffold. RESULTS: In the repair of cartilage defects in rabbit knees, NGF-SF/CS-BMSCs resulted in higher ICRS scores and lower modified Wakitani scores. The in vitro results showed that there was no significant correlation between the proliferation of BMSCs and the addition of different concentrations of NGF. Additionally, there was no significant difference in the protein and mRNA expression of COL2a1 and ACAN between the groups after the addition of different concentrations of NGF. CONCLUSION: NGF-SF/CS-BMSCs improved the repair of articular cartilage defects in the knees of rabbits. This repair effect may be related to the early promotion of subchondral bone repair.

Green Fluorescent Carbon Dots with Critically Controlled Surface States: Make Silk Shine via Feeding Silkworms.

Feeding silkworms with functional materials as additives to produce naturally modified silk is a facile, diverse, controllable, and environmentally friendly method with a low cost of time and investment. Among various additives, carbon dots (CDs) show unique advantages due to their excellent biocompatibility and fluorescence stability. Here, a new type of green fluorescent carbon dots (G-CDs) is synthesized with a high oil-water partition ratio of 147, a low isoelectric point of 5.16, an absolute quantum yield of 71%, and critically controlled surface states. After feeding with G-CDs, the silkworms weave light yellow cocoons whose green fluorescence is visible to the naked eye under UV light. The luminous silk is sewn onto the cloth to create striking patterns with beautiful fluorescence. Such G-CDs have no adverse effect on the survival rate and the life cycle of silkworms and enable their whole bodies to glow under UV light. Based on the strong fluorescence, chemical stability, and biological safety, G-CDs are found in the digestive tracts, silk glands, feces, cocoons, and even moth bodies. G-CDs accumulate in the posterior silk glands where fibroin protein is secreted, indicating its stronger combination with fibroin than sericin, which meets the requirements for practical applications.

Silk fibroin wetting stability film induced by polyamide-amine-epichlorohydrin (PAE) for intelligent sensing system.

Protein materials gain new functions and applicability through redesigns in protein structure and engineering confer. However, the application and development of proteins for use in flexible devices that fit in flexible devices that fit the surface of human skin is hindered by their poor wet stability. Here, we described the design of wet-stable materials based on the reconstruction of silk fibroin (SF). The combination of polyamide-amine-epichlorohydrin (PAE) was used as a traction rope to bring SF molecular chains closer to each other, to facilitate the self-assembly of SF through branching and lengthening of molecular chains, and change its crystalline structure. SF/PAE composite films that exhibited huge improvement in ductility and wet stability were combined with flexible SF substrates via patterning and ion sputtering to prepare flexible sensors. In addition, the SF/PAE sensing system equipped with a microprocessor and Bluetooth module enabled the real-time remote acquisition of human health signals such as vocal cords, joints, pulse and meridians. This reconfiguration of the SF structure will advance the systematic exploration of protein structures and the development of protein materials for intelligent device applications.

Magnetic-navigable silk fibroin microneedles for oral drug delivery: Ensuring long-lasting helicobacter pylori eradication and rapid hemostasis in the stomach.

The Helicobacter pylori infection in the stomach is the key reason for gastric mucosal bleeding. Eliminating gastric Helicobacter pylori by oral treatment remains difficult due to the presence of the gastric mucosal layer, which acts as a physical barrier to drugs via oral administration. In this study, a magnetic-navigable microneedle drug delivery platform (MNsD) for oral administration, featuring differential dual-mode drug release rate, was designed to fulfil rapid gastric hemostasis and overcome the gastric barriers for long-lasting Helicobacter pylori inhibition in stomach. MNs-D was created by rationally loading the carrier substrate, which was composed of silk fibroin with variable solubility, with antibiotics and hemostats. In vitro experiments showed MNs-D may sustainably eradicate Helicobacter pylori in stimulated gastric juices with long-lasting drug release (79 % in 24 h) and quickly establish hemostasis with instant drug release (92 % within 60 s). Most importantly, in vivo studies demonstrated MNs-D overcame the unsettling gastric mucosal barrier in traditional therapies of oral administration by insertion into the GML under magnetic navigation, resulting in sustained antibiotic release for long-lasting Helicobacter pylori eradiation (99 %). For differential dual-mode medication release against gastric Helicobacter pylori infections, this study may have firstly examined the effects of magnetic navigated microneedles administered orally.

Unveiling the synergy: Biocompatible alginate-cellulose hydrogel loaded with silk fibroin and zinc ferrite nanoparticles for enhanced cell adhesion, and anti-biofilm activity.

Combining a biocompatible hydrogel scaffold with the cell-supportive properties of silk fibroin (SF) and the unique functionalities of ZnFe2O4 nanoparticles creates a promising platform for advanced nanobiomaterials. The research is centered on synthesizing a natural hydrogel using cellulose (Cellul) and sodium alginate (SA) combined with SF and zinc ferrite nanoparticles. A range of analytical and biological assays were conducted to determine the biological and physicochemical properties of the nanobiocomposite. The hemolysis and 2,5-diphenyl-2H-tetrazolium bromide (MTT) assays indicated that the SA-Cellul hydrogel/SF/ZnFe2O4 nanobiocomposite was a biocompatible against human dermal fibroblasts (Hu02) and red blood cells (RBC). In addition, aside from demonstrating outstanding anti-biofilm activity, the nanobiocomposite also promotes the Hu02 cells adhesion, showcasing the synergistic effect of incorporating SF and ZnFe2O4 nanoparticle. These promising results show that this nanobiocomposite has potential applications in various biomedical fields.

Overview and Evolution of Insect Fibroin Heavy Chain (FibH).

The FibH gene, crucial for silk spinning in insects, encodes a protein that significantly influences silk fiber mechanics. Due to its large size and repetitive sequences, limited known sequences of insect FibH impede comprehensive understanding. Here, we analyzed 114 complete FibH gene sequences from Lepidoptera (71 moths, 24 butterflies) and 13 Trichoptera, revealing single-copy FibH in most species, with 2-3 copies in Hesperinae and Heteropterinae (subfamily of skippers). All FibH genes are structured with two exons and one intron (39-45 bp), with the second exon being notably longer. Moths exhibit higher GC content in FibH compared to butterflies and Trichoptera. The FibH composition varies among species, with moths and butterflies favoring Ala, Gly, Ser, Pro, Gln, and Asn, while Trichoptera FibH is enriched in Gly, Ser, and Arg, and has less Ala. Unique to Trichoptera FibH are Tyr, Val, Arg, and Trp, whereas Lepidoptera FibH is marked by polyAla (polyalanine), polySer (polyserine), and the hexapeptide GAGSGA. A phylogenetic analysis suggests that Lepidoptera FibH evolved from Trichoptera, with skipper FibH evolving from Papilionoidea. This study substantially expands the FibH repertoire, providing a foundation for the development of artificial silk.

Use of Silk Fibroin Material Composites for Green, Flexible Supercapacitors.

Within the context of seeking eco-friendly and readily available materials for energy storage, there is a pressing demand for energy storage solutions that employ environmentally sustainable, high-performance, and adaptable constituents. Specifically, such materials are essential for use in wearable technology, smart sensors, and implantable medical devices, whereas, more broadly, their use plays a pivotal role in shaping their efficiency and ecological footprint. Here, we demonstrate an entirely biopolymer-based supercapacitor with a remarkable performance, achieving a capacitance greater than 0.2 F cm-2 at a charge-discharge current of 10 mA cm-2 with 94% capacitance retention after 20,000 cycles. The supercapacitor is composed of three distinct silk fibroin (SF) composite materials, namely, photo-cross-linkable SF (Sil-MA) hydrogel, SF-polydopamine (SF-PDA), and SF bioplastic, to create a gel electrolyte, electrode binder, and encapsulation, respectively. Together, these elements form a mechanically and electrochemically robust skeleton for biofriendly energy storage devices. Moreover, these biomaterial-based supercapacitor devices show stretchability, flexibility, and compressibility while maintaining their electrochemical performance. The biomaterials and fabrication techniques presented can serve as a foundation for investigating various aqueous electrochemical energy storage systems, especially for emerging applications in wearable electronics and environmentally friendly material systems.

Curcumin-shellac nanoparticle-loaded GelMA/SilMA hydrogel for colorectal cancer therapy.

In this study, a novel approach was employed to develop a therapeutic system for colorectal cancer treatment. Specifically, a GelMA/SilMA hydrogel loaded with curcumin-shellac nanoparticles (Cur@Lac NPs) was created. A microfluidic swirl mixer was utilized to formulate stable Cur@Lac NPs, ensuring their consistent and effective encapsulation. The pH-specific release of curcumin from the NPs demonstrated their potential for colon cancer treatment. By carefully regulating the ratio of GelMA (gelatin methacrylate) and SilMA (silk fibroin methacrylate), a GelMA/SilMA dual network hydrogel was generated, offering controlled release and degradation capabilities. The incorporation of SilMA notably enhanced the mechanical properties of the dual network matrix, improving compression resistance and mitigating deformation. This mechanical improvement is crucial for maintaining the structural integrity of the hydrogel during in vivo applications. In comparison to the direct incubation of curcumin, the strategy of encapsulating curcumin into NPs and embedding them within the GelMA/SilMA hydrogel resulted in more controlled release mechanisms. This controlled release was achieved through the disintegration of the NPs and the swelling and degradation of the hydrogel matrix. The encapsulating strategy also demonstrated enhanced cellular uptake of curcumin, leveraging the advantages of both NPs and in-situ hydrogel injection. This combination ensures a more efficient and sustained delivery of the therapeutic agent directly to the tumor site. Overall, this approach holds significant promise as a smart drug delivery system, potentially improving the efficacy of colorectal cancer treatments by providing targeted, controlled, and sustained drug release with enhanced mechanical stability and biocompatibility.

In Situ ATR-FTIR Studies on the ß-Sheet Formation of Native and Regenerated Bombyx mori Silk Material in Solution and Its Potential for Drug Releasing Coatings.

Dynamic attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy at both solutions and coatings of a semicrystalline silk material derived from Bombyx mori was applied to monitor the ß-sheet conformation, which is known to correlate with silk protein crystallinity. The secondary structure-sensitive Amide I band was analyzed. Two silk protein samples were studied: native-based silk buffer fibroin (NSF) was extracted from silk glands and regenerated silk fibroin (RSF) was extracted from degummed cocoons. Solutions of both NSF and RSF at 2 mg/mL featured low initial ß-sheet contents of 5-12%, which further increased to 47-53% after 24 h. RSF and NSF solutions at 23 mg/mL also featured low initial ß-sheet contents of 9-10%, which yet only slightly increased to 16-17% after 24 h. Coatings deposited from RSF solutions showed high surface integrity (Q > 99%) after rinsing in mineralized water, enabling interfacial drug delivery applications. RSF coatings were post-treated with either formic acid (FA) or pure methanol (MeOH) vapor to showcase inducibility of crystalline domains in RSF coatings. Such coatings were loaded with the model antibiotic drugs tetracycline (TCL) and streptomycin (STRP), and the sustained release of TCL was followed in contact with (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer. RSF/TCL coatings post-treated with formic acid (FA) vapor followed by methanol (MeOH) vapor showed a significantly lower (52%) initial burst of rather hydrophobic TCL compared to untreated RSF/TCL coatings (72%), while no such significant release difference was observed for hydrophilic STRP. This was rationalized by a specific interaction between nonpolar TCL and hydrophobic crystalline RSF domains.

Permselectivity of Silk Fibroin Hydrogels for Advanced Drug Delivery Neurotherapies.

A promising trend in tissue engineering is using biomaterials to improve the control of drug concentration in targeted tissue. These vehicular systems are of specific interest when the required treatment time window is higher than the stability of therapeutic molecules in the body. Herein, the capacity of silk fibroin hydrogels to release different molecules and drugs in a sustained manner was evaluated. We found that a biomaterial format, obtained by an entirely aqueous-based process, could release molecules of variable molecular weight and charge with a preferential delivery of negatively charged molecules. Although the theoretical modeling suggested that drug delivery was more likely to be driven by Fickian diffusion, the external media had a considerable influence on the release, with lipophilic organic solvents such as acetonitrile-methanol (ACN-MeOH) intensifying the release of hydrophobic molecules. Second, we found that silk fibroin could be used as a vehicular system to treat a variety of brain disorders as this biomaterial sustained the release of different factors with neurotrophic (brain-derived neurotrophic factor) (BDNF), chemoattractant (C-X-C motif chemokine 12) (CXCL12), anti-inflammatory (TGF-ß-1), and angiogenic (VEGF) capacities. Finally, we demonstrated that this biomaterial hydrogel could release cholesteronitrone ISQ201, a nitrone with antioxidant capacity, showing neuroprotective activity in an in vitro model of ischemia-reoxygenation. Given the slow degradation rate shown by silk fibroin in many biological tissues, including the nervous system, our study expands the restricted list of drug delivery-based biomaterial systems with therapeutic capacity for both short- and especially long-term treatment windows and has merit for use with brain pathologies.