Silk Films Research Articles

Down-regulation of MYCN protein by CX-5461 leads to neuroblastoma tumor growth suppression

PurposeMYCN oncogene amplification is an independent predictor of poor prognosis in neuroblastoma. CX-5461 is a small molecular inhibitor that prevents initiation of ribosomal RNA (rRNA) synthesis by RNA Pol I, down-regulating MYCN/MYC proteins. We hypothesize that neuroblastoma tumor growth can be suppressed by CX-5461. MethodsMYCN-amplified (KELLY, IMR5) and nonamplified (SY5Y, SKNAS) neuroblastoma cells were treated with CX-5461. MYCN/MYC expression after 24–48 h was determined by Western blot. Orthotopic neuroblastoma tumors created in mice using KELLY cells were treated with CX-5461-loaded silk films implanted locally. Tumor growth was monitored using ultrasound. Histologic evaluation of tumors was performed. ResultsIC50 for KELLY, IMR5, SY5Y, and SKNAS cells to CX-5461 was 0.75 μM, 0.02 μM, 0.8 μM, and 1.7 μM, respectively. CX-5461 down-regulated MYCN and MYC proteins at 0.25–1.0 μM on Western blot analysis. CX-5461-loaded silk film released 23.7±3 μg of the drug in 24 h and 48.2±3.9 μg at 120 h. KELLY tumors treated with CX-5461-loaded film reached 800 mm3 after 7.8±1.4 days, while those treated with control film reached the same size on 5.1±0.6 days (p=0.03). CX-5461-treated tumors showed collapse of nucleolar hypertrophy and MYCN protein downregulation. ConclusionWe demonstrated that local delivery of CX-5461 via sustained release platform can suppress orthotopic neuroblastoma tumor growth, especially those with MYCN/MYC overexpression.

3,4-Dihydroxyphenylalanine (DOPA)-Containing Silk Fibroin: Its Enzymatic Synthesis and Adhesion Properties.

Silk fibroin (SF) is a fascinating natural biomaterial that exhibits remarkable mechanical properties and biocompatibility. Meanwhile, biological adhesive materials have gathered much attention as biomedical and ecofriendly materials as a result of their characteristic properties. Herein, we report the excellent adhesive function of enzymatically modified SF. The tyrosine residues of SF were successfully converted to the dihydroxy-l-phenylalanine (DOPA) unit using tyrosinase as a biocatalyst. The content of DOPA was evaluated by amino acid composition analysis. Adhesive functions of DOPA-modified SF (DOPA-SF) among several material surfaces including mica, paper, polypropylene, wood, and silk film were elucidated by lap shear tests. Fourier transform infrared measurements demonstrated that the adhesion strength of DOPA-SF was not directly related to the β-sheet formation of silk molecules. This ecofriendly and facile method offers a new perspective for fabricating natural adhesive materials for various application areas.

Water-Rinsed Nonmulberry Silk Film for Potential Tissue Engineering Applications

The possibility of using nonmulberry silk fibroin as a biomaterial is a subject of broad interest due to its inherent RGD sequence. However, the extraction of silk fibroin from cocoons and silk glands remains a formidable task. In this work, we report a facile method to dissolve tussah silk using CaCl2–formic acid and a water-rinsing process for regeneration of tussah silk fibroin (TSF) film. The new technique is important and valuable because it can directly extract silk fibroin from cocoons and is advantageous over other previous reported methods using LiSCN and Ca(NO3)2. The morphology, chemical structure, and mechanical properties of regenerated film rinsed with water, ethanol, and methanol are characterized by scanning electron microscopy, Fourier transform infrared, and X-ray diffraction techniques in combination with mechanical testing. The results reveal that the regenerated TSF films possess a smooth surface and nanostructural cross section and exhibit predominantly β-sheet crystal structure and ...

The Effect of Micro- and Nanoscale Surface Topographies on Silk on Human Corneal Limbal Epithelial Cell Differentiation

We previously reported that micro- and nano-scale topographic pitch created on silk films mimic features of the corneal basement membrane by providing biophysical cues to direct corneal epithelial cell adherence and migration. However, the effect of these topographical features on corneal limbal epithelial cell differentiation has not been explored. We hypothesize in the current study that various topographical pitch created on silk may affect corneal epithelial stem cell differentiation and alter the expression of genes involved in cell differentiation and self-renewal. We patterned silk films with different topographic pitch via soft lithography and observed human corneal limbal epithelial cell behavior. Colony forming assay demonstrated increased colony forming efficiency on patterned silk films. Cells cultured on nanoscale patterned silk films also expressed lower levels of putative keratocyte differentiation markers and higher levels of putative limbal stem cell markers. RNA-Seq analysis further implicated the involvement of pathways related to stem cell differentiation and self-renewal, including Notch, ERK/MAPK and Wnt/β-catenin signaling. We conclude that patterned silk film substrates can be used as scaffolds and provide biophysical cues to corneal limbal stem cells that may maintain corneal epithelial stem cells at a less differentiated state.

Genetically engineered bi-functional silk material with improved cell proliferation and anti-inflammatory activity for medical application

Functional silk is a promising material for future medical applications. These include fabrication of diverse silk fiber and silk protein-regenerated biomaterials such as silk sutures, hydrogel, films, and 3D scaffolds for wound healing and tissue regeneration and reconstruction. Here, a novel bi-functional silk with improved cell proliferation and anti-inflammatory activities was created by co-expressing the human basic fibroblast growth factor (FGF2) and transforming growth factor-β1 (TGF_β1) genes in silkworm. First, both FGF2 and TGF_β1 genes were confirmed to be successfully expressed in silk thread. The characterization of silk properties by SEM, FTIR, and mechanical tests showed that this new silk (FT silk) had a similar diameter, inner molecular composition, and mechanical properties as those of normal silk. Additionally, expressed FGF2 and TGF_β1 proteins were continuously and slowly released from FT silk for one week. Most importantly, the FGF2 and TGF_β1 contained in FT silk not only promoted cell proliferation by activating the ERK pathway but also significantly reduced LPS-induced inflammation responses in macrophages by mediating the Smad pathway. Moreover, this FT silk had no apparent toxicity for cell growth and caused no cell inflammation. These properties suggest that it has a potential for medical applications. Statement of SignificanceSilk spun by domestic silkworm is a promising material for fabricating various silk protein regenerated biomaterials in medical area, since it owes good biocompatibility, biodegradability and low immunogenicity. Recently, fabricating various functional silk fibers and regenerated silk protein biomaterials which has ability of releasing functional protein factor is the hot point field. This study is a first time to create a novel bi-functional silk material with the improved cell proliferation and anti-inflammatory activity by genetic engineered technology. This novel silk has a great application potential as new and novel medical material, and this study also provides a new strategy to create various functional or multifunctional silk fiber materials in future.

Development of regenerated silk films coated with fluorinated polypeptides to achieve high water repellency and biodegradability in seawater

In this study, we prepared silkworm silk films with fluorinated polypeptide coatings on their surface to achieve water-repellent silk materials. The silk films were smoothly and uniformly coated with the fluorinated polypeptides to give a transparent film. The contact angles of water droplets on the polypeptide-coated silk films were measured to investigate the ability of the films to resist water. The silk films coated with the fluorinated polypeptides exhibited higher contact angles compared to those of the uncoated silk film, indicating that the fluorinated polypeptides formed a hydrophobic surface on the silk film. The structure of the fluorinated polypeptides influenced the water-repellent ability, and the polypeptide containing both perfluoroalkyl chains and biphenyl groups imparted the highest water repellency. A biochemical oxygen demand (BOD) assay for the polypeptide-coated silk films in seawater revealed that the silk films showed high biodegradability in seawater even with the fluorinated polypeptide coating. The polypeptide-coated silk films provide not only high water repellency for practical use but also high biodegradability in marine environments.

Humidity sensing using THz metamaterial with silk protein fibroin.

In this study, we developed hybrid humidity sensing methods by incorporating silk fibroin protein onto metamaterials, operating in the terahertz (THz) frequencies; the resonant frequency shifted but saturated at a specific thickness due to the limited sensing volume of the metamaterial. From the saturated value, we extracted the dielectric constant for the silk films. We also observed additional resonance shifts when we applied humid air to silk-coated metamaterials, due to the increased water molecule numbers on the film. Frequency shifts depend linearly on relative humidity. Also, in situ THz spectroscopy measurements reveal that the time response is instantaneous within our detection limit, especially upon exposure to humid air, whereas the small slowly decaying component appeared when we applied dry air. The time taken by the slow component in the drying process was 10-50 s, depending on film thickness. This could optimize humidity sensors as a fast and efficient detection tool to measure air humidity.

Surface Patterning and Innate Physicochemical Attributes of Silk Films Concomitantly Govern Vascular Cell Dynamics.

Functional impairment of vascular cells is associated with cardiovascular pathologies. Recent literature clearly presents evidence relating cell microenvironment and their function. It is crucial to understand the cell-material interaction while designing a functional tissue engineered vascular graft. Natural silk biopolymer has shown potential for various tissue-engineering applications. In the present work, we aimed to explore the combinatorial effect of variable innate physicochemical properties and topographies of silk films on functional behavior of vascular cells. Silk proteins from different varieties (mulberry Bombyx mori, BM; and non-mulberry Antheraea assama, AA) possess unique inherent amino acid composition that leads to variable surface properties (roughness, wettability, chemistry, and mechanical stiffness). In addition, we engineered the silk film surfaces and printed a microgrooved pattern to induce unidirectional cell orientation mimicking their native form. Patterned silk films induced unidirectional alignment of porcine vascular cells. Regardless of alignment, endothelial cells (ECs) proliferated favorably on AA films; however, it suppressed production of nitric oxide (NO), an endogenous vasodilator. Unidirectional alignment of smooth muscle cells (SMCs) encouraged contractile phenotype as indicated by minimal cell proliferation, increment of quiescent (G0) phase cells, and upregulation of contractile genes. Moderately hydrophilic flat BM films induced cell aggregation and augmented the expression of contractile genes (for SMCs) and endothelial nitric oxide synthase, eNOS (for ECs). Functional studies further confirmed SMCs' alignment improving collagen production, remodeling ability (matrix metalloproteinase, MMP-2 and MMP-9 production) and physical contraction. Altogether, this study confirms vascular cells' functional behavior is crucially regulated by synergistic effect of their alignment and cell-substrate interfacial properties.

Silk-Based Antimicrobial Polymers as a New Platform to Design Drug-Free Materials to Impede Microbial Infections.

Surgical site infections (SSI) represent a serious health problem that occur after invasive surgery, thus new antimicrobial biomaterials able to prevent SSI are needed. Silks are natural biopolymers with excellent biocompatibility, low immunogenicity and controllable biodegradability. Spider silk-based materials can be bioengineered and functionalized with specific peptides, such as antimicrobial peptides, creating innovative polymers. Herein, we explored new drug-free multifunctional silk films with antimicrobial properties, specifically tailored to hamper microbial infections. Different spider silk domains derived from the dragline sequence of the spider Nephila clavipes (6mer and 15mer, 27 and 41 kDa proteins, respectively) were fused with the two antimicrobial peptides, Hepcidin (Hep) and Human Neutrophil peptide 1 (HNP1). The self-assembly features of the spider silk domains (β-sheets) were maintained after functionalization. The bioengineered 6mer-HNP1 protein demonstrated inhibitory effects against microbial pathogens. Silk-based films with 6mer-HNP1 and different contents of silk fibroin (SF) significantly reduced bacterial adhesion and biofilm formation, whereas higher bacterial counts were found on the films prepared with 6mer or SF alone. The silk-based films showed no cytotoxic effects on human foreskin fibroblasts. The positive cellular response, together with structural and antimicrobial properties, highlight the potential of these multifunctional silk-based films as new materials for preventing SSI.

Layer-by-layer self-assembled films of silk fibroin/collagen/poly (diallyldimethylammonium chloride) as nucleating surface for osseointegration to design coated dental implant materials

A coated material with a nucleating surface for osseointegration is an attractive choice for dental implant materials. Layer-by-layer (LbL) self-assembly film coating was used to fabricate a nucleating surface for osseointegration to design coated dental implant materials. Silk fibroin (SF), collagen (Col), and poly (diallyldimethylammonium chloride) (PDDA) were selected as the base materials for the films. LbL self-assembly films of PDDA-SF-PDDA-Col were constructed with different n layers: n = 0 (control), 10, 20, 30, 40, and 50. Quartz crystal microbalance, atomic force microscopy, scanning electron microscopy, and wettability testing were used to analyze the multilayer formation, topography, morphology, and surface characteristics of the films, respectively. Molecular organization of the films was characterized by Fourier transform infrared and Raman spectroscopy. The biological performance was evaluated with osteoblast cell proliferation, alkaline phosphatase (ALP) activity, and total protein absorption. The multilayer showed a linear function of thickness and the number of layers. The multilayer films demonstrated mobility of amide I, II, III, and molecular skeletal vibration. The films had rough surfaces, hydrophilicity, biological performance to enhance cell proliferation, ALP activity, and total protein absorption. The results indicated the films were promising as a nucleating surface for osseointegration in the design of coated dental implant materials.

Exploring the Structural Transformation Mechanism of Chinese and Thailand Silk Fibroin Fibers and Formic-Acid Fabricated Silk Films.

Silk fibroin (SF) is a protein polymer derived from insects, which has unique mechanical properties and tunable biodegradation rate due to its variable structures. Here, the variability of structural, thermal, and mechanical properties of two domesticated silk films (Chinese and Thailand B. Mori) regenerated from formic acid solution, as well as their original fibers, were compared and investigated using dynamic mechanical analysis (DMA) and Fourier transform infrared spectrometry (FTIR). Four relaxation events appeared clearly during the temperature region of 25 °C to 280 °C in DMA curves, and their disorder degree (fdis) and glass transition temperature (Tg) were predicted using Group Interaction Modeling (GIM). Compared with Thai (Thailand) regenerated silks, Chin (Chinese) silks possess a lower Tg, higher fdis, and better elasticity and mechanical strength. As the calcium chloride content in the initial processing solvent increases (1%–6%), the Tg of the final SF samples gradually decrease, while their fdis increase. Besides, SF with more non-crystalline structures shows high plasticity. Two α- relaxations in the glass transition region of tan δ curve were identified due to the structural transition of silk protein. These findings provide a new perspective for the design of advanced protein biomaterials with different secondary structures, and facilitate a comprehensive understanding of the structure-property relationship of various biopolymers in the future.

Self-Patterning Silk Films for Cellular Coculture.

Silk produced from Bombyx mori as a natural polymer has gained tremendous attraction for biomedical applications. Tissue engineering applications require meticulous spatial control over cellular adhesion, which is conventionally acquired via lithographic techniques. Here, we report formation of structures using nanogels that are readily cross-linked in the presence of photoactive groups that form stable, biocompatible patterned system for cellular growth. This direct approach has the proficiency to commission structures with reduced processing steps for obtaining biocompatible surface favoring stem cell adhesion, differentiation, and subsequent coculture with an additional cell type in a spatially controlled manner.

The Biomedical Use of Silk: Past, Present, Future.

Humans have long appreciated silk for its lustrous appeal and remarkable physical properties, yet as the mysteries of silk are unraveled, it becomes clear that this outstanding biopolymer is more than a high-tech fiber. This progress report provides a critical but detailed insight into the biomedical use of silk. This journey begins with a historical perspective of silk and its uses, including the long-standing desire to reverse engineer silk. Selected silk structure-function relationships are then examined to appreciate past and current silk challenges. From this, biocompatibility and biodegradation are reviewed with a specific focus of silk performance in humans. The current clinical uses of silk (e.g., sutures, surgical meshes, and fabrics) are discussed, as well as clinical trials (e.g., wound healing, tissue engineering) and emerging biomedical applications of silk across selected formats, such as silk solution, films, scaffolds, electrospun materials, hydrogels, and particles. The journey finishes with a look at the roadmap of next-generation recombinant silks, especially the development pipeline of this new industry for clinical use.

Constructing Multilayer Silk Protein/Nanosilver Biofunctionalized Hierarchically Structured 3D Printed Ti6Al4 V Scaffold for Repair of Infective Bone Defects.

Biomaterials-enabled regenerative medicine in orthopedics is challenged with infective bone defects that do not heal normally. Three-dimensional (3D) scaffold biomaterials simultaneously emulating skeletal hierarchy and eliciting sustainable osteogenetic and antibacterial functionalities represent a potent solution holding increasing fascination. Here we describe a simple combinatorial strategy for constructing such scaffolds. Fully porous titanium was first tailor-made by metallic powder 3D printing and subjected to in situ hydrothermal growth of a micro/nanostructured titanate layer, to which nanosilver encapsulated, physically cross-linked silk fibrin multilayer films were anchored through polydopamine-assisted, silk-on-silk self-assembly. The hydrophilicity, protein adsorption, and surface bioactivity of the scaffolds were investigated. Employing clinically relevant pathogenic Staphylococcus aureus bacteria, we tested that the silver immobilized scaffolds not only reduced adherence of bacteria on the surface, they also actively killed those planktonic, and these performances were largely maintained over an extended period of 6 weeks. Additionally, our engineered scaffolds were amenable to 14 days' continuous, intense bacterial attacks showing little sign of biofilm colonization, and they were interestingly capable of eradicating bacteria in already formed biofilms. High cargo loading, durable topical Ag+ release, and overwhelming oxidative stress were shown to contribute to this sustainable antibacterial system. Irrespective of certain degree of cellular stress at early stages, our scaffolds elicited generally enhanced cell proliferation, alkaline phosphatase enzyme production, and matrix calcification of osteoblastic MC3T3-E1. These multifunctionalities, coupled with the design freedom, shape flexibility, and cost-effectiveness offered by 3D printing, make our scaffold biomaterials a promising option for customized restoration of complicated infective bone defects.

"Print-to-pattern": Silk-Based Water Lithography.

The requirement of nontoxic and versatile manufacturing frameworks for biologically relevant applications has imposed significant constraints on the choice of functional materials and the complementary fabrication tools. In this context, silk is actively studied, thanks to its mechanical robustness, biocompatibility, wide availability, aqueous processing conditions, and ease of functionalization. The inherent matching between the water solubility of silk and the aqueous inks of the inkjet printing (IJP) process has derived a biofriendly and versatile "print-to-pattern" scheme-termed silk-based water lithography-toward scalable functional biomanufacturing. The deposition mode of IJP and the etching effect of silk film by water features a dual tone fabrication where functional molecules are dispensed additively, while the silk film is patterned in a "subtractive" fashion. Such versatility and scalability pave the way to a wide range of opportunities in the biomedical field.

Effect of molecular weight on the structure and mechanical properties of silk sericin gel, film, and sponge

In this study, the effect of the MW on the structure and properties of sericin film, sponge, and gel was examined. As the MW of sericin increased, the gelation of the sericin aqueous solution was found to be accelerated, and the gel strength, and the gel-sol transition temperature increased. Irrespective of the casting solvent (water and formic acid) and form of sericin (gel, film, or sponge), the crystallization of the sericins was accelerated. The mechanical properties of the sericin sponge were remarkably improved upon increasing the MW of sericin. The MW of sericin almost did not have an effect on the cell toxicity. As the MW of sericin is increased, the sericin sponge becomes denser and its porosity is reduced, leading to a decrease in the swelling ratio. These results indicate that various characteristics of the sericin forms can be modulated by controlling the MW of sericin, with enhanced potential for biomedical and cosmetic applications.

Abstract 2497: Inhibitors of RNA Pol I and an aminoacyl-tRNA synthetase result in MYC and MYCN downregulation in MYC family protein-driven neuroblastoma cells

Abstract We have previously reported histologic variants among unfavorable histology neuroblastomas (NB), having large cell appearance with vesicular nuclei and or prominent nucleolar (PN) formation, indicating hyperactive rRNA synthesis and protein translation. These features appear to be associated with dismal outcome and high-level MYC family protein expression. PN formation could provide critical prognostic and risk-stratification information for NB. At the same time, this observation could open up an opportunity for innovative therapy for aggressive NB. To assess this possibility, we examined the effect of two potent and specific small-molecule inhibitors of RNA Pol I activity and an aminoacyl tRNA synthetase (CX-5461 and Halofuginone, respectively) on growth and MYC family protein expression in NB cell lines. These small-molecule inhibitors inhibited growth of NB cell lines at low to submicromolar concentrations in vitro in 48 hours. Moreover, the inhibitors destabilized MYC and MYCN proteins in NB cells. Interestingly, halofuginone showed a rapid effect on the stability of MYC family proteins at 500 nM-1 uM (<3 hours). In contrast, CX-5461 at 1 uM was less effective at the 3-hour time. At the concentration of 100 nM-250 nM for halofuginone and 1 uM for CX-5461 for up to 48 hours of the drug treatments, MYC and MYCN expression were both downregulated. We further examined the preclinical efficacy of halofuginone in orthotopic Kelly human NB xenografts in mice using a silk film as the drug delivery device. Halofuginone showed a significant growth-suppressive effect on the xenografts, as indicated by the time periods to reach certain tumor sizes being significantly longer in the halofuginone-treated group than those in the control group. Moreover, histologic examination of the xenografts showed a marked suppression of MYCN protein expression in the halofuginone-treated xenograft at day 17 of the drug treatment. Specific inhibition of hyperactive rRNA synthesis and protein translation was shown to be an effective way to suppress MYC/MYCN protein expression and NB growth. Together, MYC-family protein overexpression and PN formation should be included in new NB risk stratification and considered for potential therapeutic targets. Citation Format: Naohiko Ikegaki, Jasmine Zeki, Bill Chiu, Hiroyuki Shimada. Inhibitors of RNA Pol I and an aminoacyl-tRNA synthetase result in MYC and MYCN downregulation in MYC family protein-driven neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2497.

Spatially controlled functional group grafting of silk films to induce osteogenic and chondrogenic differentiation of human mesenchymal stem cells

For tissue engineering, fabrication of appropriate biomaterials, which not only support cellular attachment and proliferation but also direct lineage-specific differentiation of stem cells is crucial. It is widely reported in literature that biomaterial surface chemistry modulates human mesenchymal stem cell (hMSC) differentiation in a lineage dependent manner. In recent years, natural materials such as silk have been used for hMSC culture because of its superior mechanical strength, biocompatibility, and biodegradability. We report here a simple strategy for differentiating hMSCs into two different lineages on the same surface. We have grafted functional groups such as acrylic acid and phosphates to guide the differentiation of hMSCs into chondrocytes and osteocytes respectively. Unlike other strategies, our strategy does not require growth factors and other added signals in the media and is initiated due to the difference of functional groups present on the surface. We believe this study will foster the development of effective silk based tissue engineered constructs.

Piezoelectric Micromachined Ultrasonic Transducer Using Silk Piezoelectric Thin Film

In this letter, we present for the first time, a successful fabrication and characterization of silk piezoelectric thin film-based piezoelectric micromachined ultrasonic transducer (PMUT). One of the exacting problems in the PMUT research is to find a suitable piezoelectric material to be used as the sensing and actuating element. Utilizing its innate piezoelectricity, we have used ultra-smooth silk thin film (RMS roughness = 2.84 nm, ${d}_{\textsf {33}} = \textsf {56.2}$ pm/V) as the piezoelectric element of the PMUT. The silicon membrane of the PMUT is realized by bulk micromachining. The challenging task of integrating the silk thin film above the micromachined membrane is successfully accomplished and the device functionality is demonstrated. The silk PMUT exhibited center frequency at 76.59 kHz with a fractional bandwidth of 3.18 %, when characterized in air.

Functional, RF-Trilayer Sensors for Tooth-Mounted, Wireless Monitoring of the Oral Cavity and Food Consumption.

Wearable devices have emerged as powerful tools for personalized healthcare in spite of some challenges that limit their widespread applicability as continuous monitors of physiological information. Here, a materials-based strategy to add utility to traditional dielectric sensors by developing a conformal radiofrequency (RF) construct composed of an active layer encapsulated between two reverse-facing split ring resonators is applied. These small (down to 2 mm × 2 mm) passive dielectric sensors possess enhanced sensitivity and can be further augmented by functionalization of this interlayer material. Demonstrator devices are shown where the interlayer is: (i) a porous silk film, and (ii) a modified PNIPAM hydrogel that swells with pH or temperature. In vivo use is demonstrated by adhesion of the device on tooth enamel to detect foods during human ingestion. Such sensors can be easily multiplexed and yield data-rich temporal information during the diffusion of analytes within the trilayer structure. This format could be extended to a suite of interlayer materials for sensing devices of added use and specificity.