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Amyloid β (Aβ) fibrils are found in the brain tissue of persons with Alzheimer's disease (AD), where they accumulate as plaques. One way to reduce the level of accumulation of Aβ in the brain and potentially treat AD is with Aβ-degrading enzymes such as neprilysin (NEP) and insulin-degrading enzyme (IDE). However, enzymatic responses and degradation mechanisms of Aβ fibrils (crystalline-state Aβ) have not been investigated, particularly with respect to how to avoid cytotoxicity of the degradation products to neuronal cells. Thus, insight into mechanisms of enzymatic degradation of Aβ fibrils would be instructive as a route to elucidating different structural features related to degradation and to cytotoxicity. We report mechanisms of enzymatic degradation of Aβ with cross-β structures and show the series of steps involved in the digestion of Aβ microfibrils to nanospheres or nanofilaments by protease XIV or α-chymotrypsin. respectively. These degradation products, which contained almost the same secondary structures, exhibited different cytotoxicities, indicating that relationships between nanoassembled structures and cytotoxicity of Aβ peptides are more significant than the β-sheet content. In addition, the enzymatic digestion at the Lys28 loop region linking the two β-sheets in Aβ fibrils is suggested as a key target related to cytotoxicity, a feature that can be selectively targeted on the basis of the choice of protease. [ABSTRACT FROM AUTHOR]

Engineered materials are ubiquitous throughout society and are critical to the development of modern technology, yet many current material systems are inexorably tied to widespread deterioration of ecological processes. Next-generation material systems can address goals of environmental sustainability by providing alternatives to fossil fuel-based materials and by reducing destructive extraction processes, energy costs, and accumulation of solid waste. However, development of sustainable materials faces several key challenges including investigation, processing, and architecting of new feedstocks that are often relatively mechanically weak, complex, and difficult to characterize or standardize. In this review paper, we outline a framework for examining sustainability in material systems and discuss how recent developments in modeling, machine learning, and other computational tools can aid the discovery of novel sustainable materials. We consider these through the lens of materiomics, an approach that considers material systems holistically by incorporating perspectives of all relevant scales, beginning with first-principles approaches and extending through the macroscale to consider sustainable material design from the bottom-up. We follow with an examination of how computational methods are currently applied to select examples of sustainable material development, with particular emphasis on bioinspired and biobased materials, and conclude with perspectives on opportunities and open challenges. [ABSTRACT FROM AUTHOR]

We report a new ultrathin filtration membrane prepared from silk nanofibrils (SNFs), directly exfoliated from natural Bombyx mori silk fibers to retain structure and physical properties. These membranes can be prepared with a thickness down to 40 nm with a narrow distribution of pore sizes ranging from 8 to 12 nm. Typically, 40 nm thick membranes prepared from SNFs have pure water fluxes of 13?000 L h-1 m-2 bar-1, more than 1000 times higher than most commercial ultrathin filtration membranes and comparable with the highest water flux reported previously. The commercial membranes are commonly prepared from polysulfone, poly(ether sulfone), and polyamide. The SNF-based ultrathin membranes exhibit efficient separation for dyes, proteins, and colloids of nanoparticles with at least a 64% rejection of Rhodamine B. This broad-spectrum filtration membrane would have potential utility in applications such as wastewater treatment, nanotechnology, food industry, and life sciences in part due to the protein-based membrane polymer (silk), combined with the robust mechanical and separation performance features. [ABSTRACT FROM AUTHOR]

Silk fibroin from the domesticated silkworm Bombyx mori is a naturally occurring biopolymer with charged hydrophilic terminal regions that end-cap a hydrophobic core consisting of repeating sequences of glycine, alanine, and serine residues. Taking inspiration from mussels that produce proteins rich in L-3,4-dihydroxyphenylalanine (DOPA) to adhere to a variety of organic and inorganic surfaces, the silk fibroin was functionalized with catechol groups. Silk fibroin was selected for its high molecular weight, tunable mechanical and degradation properties, aqueous processability, and wide availability. The synthesis of catechol-functionalized silk fibroin polymers containing varying amounts of hydrophilic polyethylene glycol (PEG, 5000 g/mol) side chains was carried out to balance silk hydrophobicity with PEG hydrophilicity. The efficiency of the catechol functionalization reaction did not vary with PEG conjugation over the range studied, although tuning the amount of PEG conjugated was essential for aqueous solubility. Adhesive bonding and cell compatibility of the resulting materials were investigated, where it was found that incorporating as little as 6 wt % PEG prior to catechol functionalization resulted in complete aqueous solubility of the catechol conjugates and increased adhesive strength compared with silk lacking catechol functionalization. Furthermore, PEG-silk fibroin conjugates maintained their ability to form ß-sheet secondary structures, which can be exploited to reduce swelling. Human mesenchymal stem cells (hMSCs) proliferated on the silks, regardless of PEG and catechol conjugation. These materials represent a protein-based approach to catechol-based adhesives, which we envision may find applicability as biodegradable adhesives and sealants. [ABSTRACT FROM AUTHOR]

Changes in expression and dysfunctional signaling of TrkA/B/C receptors and oncogenic Trk fusion proteins are found in neurological diseases and cancers. Here, we describe the development of a first 18F-labeled optimized lead suitable for in vivo imaging of Trk, [18F]TRACK, which is radiosynthesized with ease from a nonactivated aryl precursor concurrently combining largely reduced P-gp liability and improved brain kinetics compared to previous leads while displaying high on-target affinity and human kinome selectivity. [ABSTRACT FROM AUTHOR]

Preliminary studies have shown that silk fibroin can protect biomacromolecules from thermal degradation, but a deeper understanding of underlying mechanisms needed to fully leverage the stabilizing potential of this matrix has not been realized. In this study, we investigate stabilization of plasma C-reactive protein (CRP), a diagnostic indicator of infection or inflammation, to gain insight into stabilizing mechanisms of silk. We observed that the addition of antiplasticizing excipients that suppress β-relaxation amplitudes in silk matrices resulted in enhanced stability of plasma CRP. These observations are consistent with those made in sugar-glass-based protein-stabilizing matrices and suggest fundamental insight into mechanisms as well as practical strategies to employ with silk protein matrices for enhanced stabilization utility. [ABSTRACT FROM AUTHOR]

The interfacial shear strength between different layers in multilayered structures of layer-by-layer (LbL) microcapsules is a crucial mechanical property to ensure their robustness. In this work, we investigated the interfacial shear strength of modified silk fibroin ionomers utilized in LbL shells, an ionic-cationic pair with complementary ionic pairing, (SF)-poly-l-glutamic acid (Glu) and SF-poly-l-lysine (Lys), and a complementary pair with partially screened Coulombic interactions due to the presence of poly(ethylene glycol) (PEG) segments and SF-Glu/SF-Lys[PEG] pair. Shearing and adhesive behavior between these silk ionomer surfaces in the swollen state were probed at different spatial scales and pressure ranges by using functionalized atomic force microscopy (AFM) tips as well as functionalized colloidal probes. The results show that both approaches were consistent in analyzing the interfacial shear strength of LbL silk ionomers at different spatial scales from a nanoscale to a fraction of a micron. Surprisingly, the interfacial shear strength between SF-Glu and SF-Lys[PEG] pair with partially screened ionic pairing was greater than the interfacial shear strength of the SF-Glu and SF-Lys pair with a high density of complementary ionic groups. The difference in interfacial shear strength and adhesive strength is suggested to be predominantly facilitated by the interlayer hydrogen bonding of complementary amino acids and overlap of highly swollen PEG segments. [ABSTRACT FROM AUTHOR]

The proto-oncogenes NTRK1/2/3 encode the tropomyosin receptor kinases TrkA/B/C which play pivotal roles in neurobiology and cancer. We describe herein the discovery of [11C]-(R)-3 ([11C]-(R)-IPMICF16), a first-in-class positron emission tomography (PET) TrkB/C-targeting radiolabeled kinase inhibitor lead. Relying on extensive human kinome vetting, we show that (R)-3 is the most potent and most selective TrkB/C inhibitor characterized to date. It is demonstrated that [11C]-(R)-3 readily crosses the blood-brain barrier (BBB) in rodents and selectively binds to TrkB/C receptors in vivo, as evidenced by entrectinib blocking studies. Substantial TrkB/C-specific binding in human brain tissue is observed in vitro, with specific reduction in the hippocampus of Alzheimer's disease (AD) versus healthy brains. We additionally provide preliminary translational data regarding the brain disposition of [11C]-(R)-3 in primates including first-in-human assessment. These results illustrate for the first time the use of a kinome-wide selective radioactive chemical probe for endogenous kinase PET neuroimaging in human. [ABSTRACT FROM AUTHOR]

Detailed control of nano- and microstructures in porous biomaterial scaffold systems is important for control of interfacial and biological functions. Self-assembled silk protein nanostructured building blocks were incorporated into salt-leached scaffolds to control these features. Controllable concentration and pH were used to induce the formation of amorphous silk nanofibers in solution and also to reduce β-sheet transformation during the more traditional salt-leaching process. These new scaffolds showed nanofibrous-microporous structures, reduced ß-sheet content, and tunable mechanical properties. Bone marrow mesenchymal stem cells grew better and showed differentiation behavior on these nanofibrous scaffolds, suggesting cytocompatibility and support for tunable differentiation via the scaffolds. These results suggested a new strategy of designing bioactive silk scaffolds by combining traditional scaffold formation processes with the controllable self-assembly of silk. [ABSTRACT FROM AUTHOR]

Conspectus: Tailored biomaterials with tunable functional properties are crucial for a variety of task-specific applications ranging from healthcare to sustainable, novel bio-nanodevices. To generate polymeric materials with predictive functional outcomes, exploiting designs from nature while morphing them toward non-natural systems offers an important strategy. Silks are Nature's building blocks and are produced by arthropods for a variety of uses that are essential for their survival. Due to the genetic control of encoded protein sequence, mechanical properties, biocompatibility, and biodegradability, silk proteins have been selected as prototype models to emulate for the tunable designs of biomaterial systems. The bottom up strategy of material design opens important opportunities to create predictive functional outcomes, following the exquisite polymeric templates inspired by silks. Recombinant DNA technology provides a systematic approach to recapitulate, vary, and evaluate the core structure peptide motifs in silks and then biosynthesize silk-based polymers by design. Post-biosynthesis processing allows for another dimension of material design by controlled or assisted assembly. Multiscale modeling, from the theoretical prospective, provides strategies to explore interactions at different length scales, leading to selective material properties. Synergy among experimental and modeling approaches can provide new and more rapid insights into the most appropriate structure-function relationships to pursue while also furthering our understanding in terms of the range of silk-based systems that can be generated. This approach utilizes nature as a blueprint for initial polymer designs with useful functions (e.g., silk fibers) but also employs modeling-guided experiments to expand the initial polymer designs into new domains of functional materials that do not exist in nature. The overall path to these new functional outcomes is greatly accelerated via the integration of modeling with experiment. In this Account, we summarize recent advances in understanding and functionalization of silk-based protein systems, with a focus on the integration of simulation and experiment for biopolymer design. Spider silk was selected as an exemplary protein to address the fundamental challenges in polymer designs, including specific insights into the role of molecular weight, hydrophobic/hydrophilic partitioning, and shear stress for silk fiber formation. To expand current silk designs toward biointerfaces and stimuli responsive materials, peptide modules from other natural proteins were added to silk designs to introduce new functions, exploiting the modular nature of silk proteins and fibrous proteins in general. The integrated approaches explored suggest that protein folding, silk volume fraction, and protein amino acid sequence changes (e.g., mutations) are critical factors for functional biomaterial designs. In summary, the integrated modeling-experimental approach described in this Account suggests a more rationally directed and more rapid method for the design of polymeric materials. It is expected that this combined use of experimental and computational approaches has a broad applicability not only for silk-based systems, but also for other polymer and composite materials. [ABSTRACT FROM AUTHOR]

Native silk fibers exhibit strength and toughness that rival those of the best synthetic fibers. Despite significant research, further insight is still needed to understand the mechanisms by which silkworms are capable of spinning such tough fibers. Here we propose that π-π and π-OH group interactions of tyrosine side chains provide templating effects, such that the crystal-forming domains are in registration, thereby fostering the self-assembly of the spinning dope. Intrinsic fluorescence measurements, in conjunction with circular dichroism, showed that during self-assembly of regenerated silk solutions, the tyrosine residues were localized in a more hydrophobic local environment, suggesting preferential assembly. In situ Fourier transform infrared spectroscopy indicated that cross-linking of the tyrosine residues resulted in the development of extended β-sheet structure. Additionally, control of cross-link density directly influenced the degree of crystallinity upon drying. Molecular dynamics simulations were performed on silk mimetic peptides in order to more thoroughly understand the role of tyrosines. The results indicated that tyrosine residues tended to transiently colocate in solution due to π-π interactions and hydrogen bonds with adjacent residues and with the peptide backbone. These more stable tyrosine interactions resulted in reduced lateral chain fluctuations and increased incidence of coordinated intrachain association, while introduction of a dityrosine bond directly promoted the formation of β-sheet structures. In total, the experimental and modeling data support a critical role for tyrosine-tyrosine interactions as a key early feature in the self-assembly of regenerated silk protein chains and therefore are important in the robust and unusual mechanical properties ultimately achieved in the process. [ABSTRACT FROM AUTHOR]

Protein hydrogels are an important class of materials for applications in biotechnology and medicine. The fine-tuning of their sequence, molecular weight, and stereochemistry offers unique opportunities to engineer biofunctionality, biocompatibility, and biodegradability into these materials. Here we report a new family of redox-sensitive protein hydrogels with controllable mechanical properties composed of recombinant silk-elastin-like protein polymers (SELPs). The SELPs were designed and synthesized with different ratios of silk-to-elastin blocks that incorporated periodic cysteine residues. The cysteine-containing SELPs were thermally responsive in solution and rapidly formed hydrogels at body temperature under physiologically relevant, mild oxidative conditions. Upon addition of a low concentration of hydrogen peroxide at 0.05% (w/v), gelation occurred within minutes for the SELPs with a protein concentration of approximately 4% (w/v). The gelation time and mechanical properties of the hydrogels were dependent on the ratio of silk to elastin. These polymer designs also significantly affected redox-sensitive release of a highly polar model drug from the hydrogels in vitro. Furthermore, oxidative gelation was performed at other physiologically relevant temperatures, and this resulted in hydrogels with tunable mechanical properties, thus, providing a secondary level of control over hydrogel stiffness. These newly developed injectable SELP hydrogels with redox-sensitive features and tunable mechanical properties may be potentially useful as biomaterials with broad applications in controlled drug delivery and tissue engineering. [ABSTRACT FROM AUTHOR]

As a functional material, silk has been widely used in tissue engineering, drug release, and tissue regeneration. Increasing subtle control of silk hierarchical structures and thus specific functional performance is required for these applications but remains a challenge. Here, we report a novel silk nanofiber solution achieved through tuning solvent systems used to generate the material. Unlike the β-sheet rich silk nanofibers reported previously, these new silk nanofibers are mainly composed of amorphous structures and maintain a solution state in aqueous environments. The amorphous silk nanofibers are stable enough for storage and also metastable, making them easy to use in the further fabrication of materials through various processes. Silk scaffolds, hydrogels, and films were prepared from these silk nanofiber solutions. These silk materials from amorphous nanofiber solutions show different properties and tunable performance features. Therefore, these amorphous silk nanofibers are suitable units or building blocks for designing silk-based materials. [ABSTRACT FROM AUTHOR]

Bombyx mori(BM) silk fibroin iscomposed of two different subunits: heavy chain and light chain fibroinlinked by a covalent disulfide bond. Current methods of separatingthe two silk fractions is complicated and produces inadequate quantitiesof the isolated components for the study of the individual light andheavy chain silks with respect to new materials. We report a simplemethod of separating silk fractions using formic acid. The formicacid treatment partially releases predominately the light chain fragment(soluble fraction) and then the soluble fraction and insoluble fractionscan be converted into new materials. The regenerated original (total)silk fibroin and the separated fractions (soluble vs insoluble) haddifferent molecular weights and showed distinctive pH stabilitiesagainst aggregation/precipitation based on particle charging. Allsilk fractions could be electrospun to give fiber mats with viscosityof the regenerated fractions being the controlling factor for successfulelectrospinning. The silk fractions could be mixed to give blendswith different proportions of the two fractions to modify the diameterand uniformity of the electrospun fibers formed. The soluble fractioncontaining the light chain was able to modify the viscosity by thinningthe insoluble fraction containing heavy chain fragments, perhaps analogousto its role in natural fiber formation where the light chain providesincreased mobility and the heavy chain producing shear thickeningeffects. The simplicity of this new separation method should enableaccess to these different silk protein fractions and accelerate theidentification of methods, modifications, and potential applicationsof these materials in biomedical and industrial applications. [ABSTRACT FROM AUTHOR]

Fibrous protein secondary structuraltransitions are affected bybound water. A family of specially designed recombinant spider silk-likeblock copolymers with a gradient of block length, hydrophobilicity,and degree of crystallinity was biosynthesized and characterized todemonstrate the effect of water on the protein structural transitions.These proteins were inspired by the genetic sequences found in thedragline silk of Nephila clavipes, comprising analanine-rich hydrophobic block, A, a glycine-rich hydrophilic block,B, and a C-terminus or a His-tag, H. Because the A-block is hydrophobicand the B-block is hydrophilic, the spider silk-like block copolymersbehave as amphiphilic molecules and self-assemble into various structuresin water solution. We employ time-resolved Fourier transform infrared(FTIR) spectroscopy to assign the origin of specific secondary structuraltransitions during heating. A transition from random coils to β-turnsdominates during a lower temperature glass transition (of plasticizedprotein) mediated by the removal of bound water. Once the proteinis in the dry solid state, further heating causes the now-dry proteinto undergo the glass transition to the liquid state through conversionof α-helices into β-turns. The structural transitionsduring protein glass transitions are intrinsic to the amorphous regionof protein and are hardly affected by protein hydrophobicity, blocklength, or crystallinity. The self-assembly morphology of the spidersilk-like block copolymers, investigated by scanning electron microscopy,indicates that the large-scale morphology is stable during heatingthrough both the lower and upper temperature glass transitions. [ABSTRACT FROM AUTHOR]

We have developed a methodology usingadvanced thermal analysisto characterize the role of water in a specially synthesized familyof recombinant spider silk-like block copolymers. These proteins wereinspired by the genetic sequences found in the dragline silk of Nephila clavipes, comprising an alanine-rich hydrophobicblock, A; a glycine-rich hydrophilic block, B; and a C-terminus ora His-tag, H. This family of proteins serves as a model system inwhich the hydrophobicity is controlled by A and B block lengths, allowingsystematic comparison of water effects within the family. Temperature-modulateddifferential scanning calorimetry and thermogravimetric analyses wereemployed to capture the glass to rubber transition, Tg, in water-cast protein films. Modeling of the solidand liquid state heat capacity baselines allows us to determine thecritical role played by bound water which plasticizes and stabilizesthe protein through interchain bonding. In samples containing boundwater, two sequential glass transitions, Tg(1) and Tg(2), were observed during heating.The lower temperature glass transition, Tg(1), is related to conformational change induced by bound water removal,the hydrophobicity of the protein sequences, and the crystallinityof the protein. The higher temperature glass transition, Tg(2), is characteristic of the dry protein. The bindingenergy of water to protein compares favorably to ligand–waterbinding affinities. The energy absorbed by evaporating water dependsupon the volume fraction of the hydrophilic B-block. [ABSTRACT FROM AUTHOR]

Silkwormsilk has been widely used as a textile fiber, as biomaterialsand in optically functional materials due to its extraordinary properties.The β-sheet-rich natural nanofiber units of about 10–50nm in diameter are often considered the origin of these properties,yet it remains unclear how silk self-assembles into these hierarchicalstructures. A new system composed of β-sheet-rich silk nanofibersabout 10–20 nm in diameter is reported here, where these nanofibersformed into “flowing hydrogels” at 0.5–2% solutionsand could be transformed back into the solution state at lower concentrations,even with a high β-sheet content. This is in contrast with othersilk processed materials, where significant β-sheet contentnegates reversibility between solution and solid states. These fibersare formed by regulating the self-assembly process of silk in aqueoussolution, which changes the distribution of negative charges whilestill supporting β-sheet formation in the structures. Mechanistically,there appears to be a shift toward negative charges along the outsideof the silk nanofibers in our present study, resulting in a higherzeta potential (above −50 mV) than previous silk materialswhich tend to be below −30 mV. The higher negative charge onsilk nanofibers resulted in electrostatic repulsion strong enoughto negate further assembly of the nanofibers. Changing silk concentrationchanged the balance between hydrophobic interactions and electrostaticrepulsion of β-sheet-rich silk nanofibers, resulting in reversiblehydrogel–solution transitions. Furthermore, the silk nanofiberscould be disassembled into shorter fibers and even nanoparticles uponultrasonic treatment following the transition from hydrogel to solutiondue to the increased dispersion of hydrophobic smaller particles,without the loss of β-sheet content, and with retention of theability to transition between hydrogel and solution states throughreversion to longer nanofibers during self-assembly. These reversiblesolution-hydrogel transitions were tunable with ultrasonic intensity,time, or temperature. [ABSTRACT FROM AUTHOR]

The design and developmentof future molecular photonic/electronicsystems pose the challenge of integrating functional molecular buildingblocks in a controlled, tunable, and reproducible manner. The modularnature and fidelity of the biosynthesis method provides a unique chemistryapproach to one-pot synthesis of environmental factor-responsive chimericproteins capable of energy conversion between the desired forms. Inthis work, facile tuning of dynamic thermal response in plasmonicnanoparticles was facilitated by genetic engineering of the structure,size, and self-assembly of the shell silk-elastin-like protein polymers(SELPs). Recombinant DNA techniques were implemented to synthesizea new family of SELPs, S4E8Gs, with amino acid repeats of [(GVGVP)4(GGGVP)(GVGVP)3(GAGAGS)4] and tunablemolecular weight. The temperature-reversible conformational switchingbetween the hydrophilic random coils and the hydrophobic β-turnsin the elastin blocks were programmed to between 50 and 60 °Cby site-specific glycine mutation, as confirmed by variable-temperatureproton NMR and circular dichroism (CD) spectroscopy, to trigger thenanoparticle aggregation. The dynamic self-aggregation/disaggregationof the Au-SELPs nanoparticles was regulated in size and pattern bythe β-sheet-forming, thermally stable silk blocks, as revealedby transmission electron microscopy (TEM) and dynamic light scattering(DLS). The thermally reversible, shell dimension dependent, interparticleplasmon coupling was investigated by both variable-temperature UV–visspectroscopy and finite-difference time-domain (FDTD)-based simulations.Good agreement between the calculated and measured spectra sheds lighton design and synthesis of responsive plasmonic nanostructures byindependently tuning the refractive index and size of the SELPs throughgenetic engineering. [ABSTRACT FROM AUTHOR]

An inkjet printing approach is presentedfor the facile fabricationof microscopic arrays of biocompatible silk “nests”capable of hosting live cells for prospective biosensors. The patterningof silk fibroin nests were constructed by the layer-by-layer (LbL)assembly of silk polyelectrolytes chemically modified with poly-(l-lysine) and poly-(l-glutamic acid) side chains. Theinkjet-printed silk circular regions with a characteristic “nest”shape had diameters of 70–100 μm and a thickness severalhundred nanometers were stabilized by ionic pairing and by the formationof the silk II crystalline secondary structure. These “locked-in”silk nests remained anchored to the substrate during incubation incell growth media to provide a biotemplated platform for printing-in,immobilization, encapsulation and growth of cells. The process ofinkjet-assisted printing is versatile and can be applied on any typeof substrate, including rigid and flexible, with scalability and facileformation. [ABSTRACT FROM AUTHOR]

Silk-elastin-likeprotein polymers (SELPs) combine the mechanicaland biological properties of silk and elastin. These properties haveled to the development of various SELP-based materials for drug delivery.However, SELPs have rarely been developed into nanoparticles, partiallydue to the complicated fabrication procedures, nor assessed for potentialas an anticancer drug delivery system. We have recently constructeda series of SELPs (SE8Y, S2E8Y, and S4E8Y) with various ratios ofsilk to elastin blocks and described their capacity to form micellar-likenanoparticles upon thermal triggering. In this study, we demonstratethat doxorubicin, a hydrophobic antitumor drug, can efficiently triggerthe self-assembly of SE8Y (SELPs with silk to elastin ratio of 1:8)into uniform micellar-like nanoparticles. The drug can be loaded inthe SE8Y nanoparticles with an efficiency around 6.5% (65 ng doxorubicin/μgSE8Y), S2E8Y with 6%, and S4E8Y with 4%, respectively. In vitro studieswith HeLa cell lines demonstrate that the protein polymers are notcytotoxic (IC50> 200 μg/mL), while the doxorubicin-loadedSE8Y nanoparticles showed a 1.8-fold higher cytotoxicity than thefree drug. Confocal laser scanning microscopy (CLSM) and flow cytometryindicate significant uptake of the SE8Y nanoparticles by the cellsand suggest internalization of the nanoparticles through endocytosis.This study provides an all-aqueous, facile method to prepare nanoscale,drug-loaded SELPs packages with potential for tumor cell treatments. [ABSTRACT FROM AUTHOR]

In this study, we utilize plasma-enhancedchemical vapor deposition(PECVD) for the deposition of nanostructures composed of diphenylalanine.PECVD is a solvent-free approach and allows sublimation of the peptideto form dense, uniform arrays of peptide nanostructures on a varietyof substrates. The PECVD deposited d-diphenylalanine nanostructureshave a range of chemical and physical properties depending on thespecific discharge parameters used during the deposition process. [ABSTRACT FROM AUTHOR]

Derived from Bombyxmoricocoons,regenerated silk fibroin (RSF) exhibits excellent biocompatibility,high toughness, and tailorable biodegradability. Additionally, RSFmaterials are flexible, optically clear, easily patterned with nanoscalefeatures, and may be doped with a variety bioactive species. Thisunique combination of properties has led to increased interest inthe use of RSF in sustainable and biocompatible electronic devices.In order to explore the applicability of this biopolymer to the developmentof future bioelectronics, the dielectric breakdown strength (Ebd) of RSF thin films was quantified as a functionof protein conformation. The application of processing conditionsthat increased β-sheet content (as determined by FTIR analysis)and produced films in the silk II structure resulted in RSF materialswith improved Ebdwith values reachingup to 400 V/μm. [ABSTRACT FROM AUTHOR]

Electrochemically controlled, reversibleassembly of biopolymersinto hydrogel structures is a promising technique for on-demand cellor drug encapsulation and release systems. An electrochemically sol–geltransition has been demonstrated in regenerated Bombyxmorisilk fibroin, offering a controllable way togenerate biocompatible and reversible adhesives and other biomedicalmaterials. Despite the involvement of an electrochemically triggeredelectrophoretic migration of the silk molecules, the mechanism ofthe reversible electrogelation remains unclear. It is, however, knownthat the freshly prepared silk electrogels (e-gels)adopt a predominantly random coil conformation, indicating a lackof cross-linking as well as thermal, mechanical, and morphologicalstabilities. In the present work, the tuning of covalent and physicalβ-sheet cross-links in silk hydrogels was studied for programmingthe structural properties. Scanning electron microscopy (SEM) revealeddelicate morphology, including locally aligned fibrillar structures,in silk e-gels, preserved by combining glutaraldehyde-cross-linkingand ethanol dehydration. Fourier transform infrared (FTIR) spectroscopicanalysis of either electrogelled, vortex-induced or spontaneouslyformed silk hydrogels showed that the secondary structure of silk e-gels was tunable between non-β-sheet-dominated andβ-sheet-dominated states. Dynamic oscillatory rheology confirmedthe mechanical reinforcement of silk e-gels providedby controlled chemical and physical cross-links. The selective incorporationof either chemical or physical or both cross-links into the electrochemicallyresponsive, originally unstructured silk e-gel shouldhelp in the design for electrochemically responsive protein polymers. [ABSTRACT FROM AUTHOR]

The recent increase in the developmentof polymer-based technologicaldevices has necessitated a surge in associated fabrication strategies.An adequate understanding of the interfacial properties in such systemsis required to meet the challenges inherent to these applications,ranging from electronics to biomedicine. In this work, we utilizethermal reflow to investigate the interfacial properties of multilayersilk fibroin film constructs. We demonstrate that reflow can be utilizedto control the water content, glass transition, and β sheetcrystallinity of such constructs, leading to control of the mechanicalproperties at the interface. Such analysis may lend insight into theinterfacial properties of similar semicrystalline biopolymers, increasingthe number of fabrication options for the development of devices atthe biological–technological nexus. [ABSTRACT FROM AUTHOR]

Ceramicscaffolds such as biphasic calcium phosphate (BCP) havebeen widely studied and used for bone regeneration, but their brittlenessand low mechanical strength are major drawbacks. We report the firstsystematic study on the effect of silk coating in improving the mechanicaland biological properties of BCP scaffolds, including (1) optimizationof the silk coating process by investigating multiple coatings, and(2) in vitro evaluation of the osteogenic response of human mesenchymalstem cells (hMSCs) on the coated scaffolds. Our results show thatmultiple silk coatings on BCP ceramic scaffolds can achieve a significantcoating effect to approach the mechanical properties of native bonetissue and positively influence osteogenesis by hMSCs over an extendedperiod. The silk coating method developed in this study representsa simple yet effective means of reinforcement that can be appliedto other types of ceramic scaffolds with similar microstructure toimprove osteogenic outcomes. [ABSTRACT FROM AUTHOR]

Conventionalfabrication techniques and structures employed inthe design of silk fibroin (SF) based porous materials provide onlylimited control over pore size and require several processing stages.In this study, it is shown that, by utilizing electrohydrodynamicbubbling, not only can new hollow spherical structures of SF be formedin a single step by means of bubbles, but the resulting bubbles canserve as pore generators when dehydrated. The bubble characteristicscan be controlled through simple adjustments to the processing parameters.Bubbles with diameters in the range of 240–1000 μm werefabricated in controlled fashion. FT-IR characterization confirmedthat the rate of air infused during processing enhanced β-sheetpacking in SF at higher flow rates. Dynamic mechanical analysis alsodemonstrated a correlation between air flow rate and film tensilestrength. Results indicate that electrohydrodynamically generatedSF and their composite bubbles can be employed as new tools to generateporous structures in a controlled manner with a range of potentialapplications in biocoatings and tissue engineering scaffolds. [ABSTRACT FROM AUTHOR]

The chimeric proteins, silk-elastin-like protein polymers (SELPs), consist of repeating units of silk and elastin to retain the mechanical strength of silk, while incorporating the dynamic environmental sensitivity of elastin. A retinal-modified SELP was prepared, modified, and studied for photodynamic responses. The protein was designed, cloned, expressed, and purified with lysine present in the elastin repeats. The purified protein was then chemically modified with the biocompatible moiety retinal via the lysine side chains. Structural changes with the polymer were assessed before and after retinal modification using Fourier transform infrared spectroscopy and circular dichroism spectroscopy. Optical studies and spectral analysis were performed before and after retinal modification. The random-coil fraction of the protein increased after retinal modification while the β-sheet fraction significantly decreased. Birefringence of the modified protein was induced when irradiated with a linearly polarized 488 nm laser light. Retinal modification of this protein offers a useful strategy for potential use in biosensors, controlled drug delivery, and other areas of biomedical engineering. [ABSTRACT FROM AUTHOR]

Substrate mechanical properties have remarkable influenceson cellbehavior and tissue regeneration. Although salt-leached silk scaffoldshave been used in tissue engineering, applications in softer tissueregeneration can be encumbered with excessive stiffness. In the presentstudy, silk-bound water interactions were regulated by controllingprocessing to allow the preparation of salt-leached porous scaffoldswith tunable mechanical properties. Increasing silk-bound water interactionsresulted in reduced silk II (β-sheet crystal) formation duringsalt-leaching, which resulted in a modulus decrease in the scaffolds.The microstructures as well as degradation behavior were also changed,implying that this water control and salt-leaching approach can beused to achieve tunable mechanical properties. Considering the utilityof silk in various fields of biomedicine, the results point to a newapproach to generate silk scaffolds with controllable properties tobetter mimic soft tissues by combining scaffold preparation methodsand silk self-assembly in aqueous solutions. [ABSTRACT FROM AUTHOR]

We studied the pH-responsive behavior of layer-by-layer(LbL) microcapsulesfabricated from silk fibroin chemically modified with different polyamino acid side chains: cationic (silk-poly l-lysine, SF-PL)or anionic (silk-poly-l-glutamic acid, SF-PG). We observedthat stable ultrathin shell microcapsules can be assembled with adramatic increase in swelling, thickness, and microroughness at extremelyacidic (pH < 2.5) and basic (pH > 11.0) conditions without noticeabledisintegration. These changes are accompanied by dramatic changesin shell permeability with a 2 orders of magnitude increase in thediffusion coefficient. Moreover, the silk ionomer shells undergo remarkablesoftening with a drop in Young’s modulus by more than 1 orderof magnitude due to the swelling, stretching, and increase in materialporosity. The ability to control permeability and mechanical propertiesover a wide range for thesilk-based microcapsules, with distinguishing stability under harshenvironmental conditions, provides an important system for controlledloading and release and applications in bioengineering. [ABSTRACT FROM AUTHOR]

The aim of this study was to understand the structureand biodegradationrelationships of silk particles intended for targeted biomedical applications.Such a study is also useful in understanding structural remodellingof silk debris that may be generated from silk-based implants. Ultrafinesilk particles were prepared using a combination of efficient wet-millingand spray-drying processes with no addition of chemicals other thanthose used in degumming. Milling reduced the intermolecular stackingforces within the β-sheet crystallites without changing theintramolecular binding energy. Because of the rough morphology andthe ultrafine size of the particles, degradation of silk particlesby protease XIV was increased by about 3-fold compared to silk fibers.Upon biodegradation, the thermal degradation temperature of silk increased,which was attributed to the formation of tight aggregates by the hydrolyzedresidual macromolecules. A model of the biodegradation mechanism ofsilk particles was developed based on the experimental data. The modelexplains the process of disintegration of β-sheets, supportedby quantitative secondary structural analysis and microscopic images. [ABSTRACT FROM AUTHOR]

Neutron reflectivity (NR) measurements of ultrathin surfacefilms(below 30 nm) composed of Bombyx morisilk fibroin protein in combination with atomic force microscopyand ellipsometry were used to reveal the internal structural organizationin both dry and swollen states. Reconstituted aqueous silk solutiondeposited on a silicon substrate using the spin-assisted layer-by-layer(SA-LbL) technique resulted in a monolayer silk film composed of randomnanofibrils with constant scattering length density (SLD). However,a vertically segregated ordering with two different regions has beenobserved in dry, thicker, seven-layer SA-LbL silk films. The verticalsegregation of silk multilayer films indicates the presence of a differentsecondary structure of silk in direct contact with the silicon oxidesurface (first 6 nm). The layered structure can be attributed to interfacialβ-sheet crystallization and the formation of well-developednanofibrillar nanoporous morphology for the initially deposited silksurface layers with the preservation of less dense, random coil secondarystructure for the layers that follow. This segregated structure ofsolid silk films defines their complex nonuniform behavior in theD2O environment with thicker silk films undergoing delaminationduring swelling. For a silk monolayer with an initial thickness of6 nm, we observed the increase in the effective thickness by 60% combinedwith surprising decrease in density. Considering the nanoporous morphologyof the hydrophobic silk layer, we suggested that the apparent increasein its thickness in liquid environment is caused by the air nanobubbletrapping phenomenon at the liquid–solid interface. [ABSTRACT FROM AUTHOR]

Organophosphates are some of the most acutely toxic compoundssynthesizedon an industrial scale, and organophosphorus hydrolase (OPH) has theability to hydrolyze and inactivate a number of these chemicals. However,OPH activity is vulnerable to harsh environmental conditions thatwould accompany its practical utility in the field; a limitation thatcan also be extended to conditions required for incorporation of OPHinto useful materials. Here we present evidence that entrapment ofOPH in silk fibroin leads to stabilization of OPH activity under avariety of conditions that would otherwise reduce free enzyme activity,such as elevated temperature, UV light exposure and the presence ofdetergent. Silk fibroin entrapment of OPH also allowed for its dispersalinto a polyurethane-based coating that retained organophosphate hydrolysisactivity after formulation, application and drying. Together, thedata presented here demonstrate the utility of silk fibroin entrapmentfor the protection of OPH activity under a variety of environmentalconditions. [ABSTRACT FROM AUTHOR]

Although natural silk fibers have excellent strengthand flexibility,the regenerated silk materials generally become brittle in the drystate. How to reconstruct the flexibility for silk fibroin has bewilderedscientists for many years. In the present study, the flexible regeneratedsilk fibroin films were achieved by simulating the natural formingand spinning process. Silk fibroin films composed of silk I structurewere first prepared by slow drying process. Then, the silk fibroinfilms were stretched in the wet state, following the structural transitionfrom silk I to silk II. The difference between the flexible film anddifferent brittle regenerated films was investigated to reveal thecritical factors in regulating the flexibility of regenerated silkmaterials. Compared with the methanol-treated silk films, althoughhaving similar silk II structure and water content, the flexible silkfilms contained more bound water rather than free water, implyingthe great influence of bound water on the flexibility. Then, furtherstudies revealed that the distribution of bound water was also a criticalfactor in improving silk flexibility in the dry state, which couldbe regulated by the nanoassembly of silk fibroin. Importantly, theresults further elucidate the relation between mechanical propertiesand silk fibroin structures, pointing to a new mode of generatingnew types of silk materials with enhanced mechanical properties inthe dry state, which would facilitate the fabrication and applicationof regenerated silk fibroin materials in different fields. [ABSTRACT FROM AUTHOR]

Silkworms and spiders generate fibers that exhibit highstrengthand extensibility. The underlying mechanisms involved in processingsilk proteins into fiber form remain incompletely understood, resultingin the failure to fully recapitulate the remarkable properties ofnative fibers in vitro from regenerated silk solutions. In the presentstudy, the extensibility and high strength of regenerated silks wereachieved by mimicking the natural spinning process. Conformationaltransitions inside micelles, followed by aggregation of micelles andtheir stabilization as they relate to the metastable structure ofsilk are described. Subsequently, the mechanisms to control the formationof nanofibrous structures were elucidated. The results clarify thatthe self-assembly of silk in aqueous solution is a thermodynamicallydriven process where kinetics also play a key role. Four key factors,molecular mobility, charge, hydrophilic interactions, and concentrationunderlie the process. Adjusting these factors can balance nanostructureand conformational composition, and be used to achieve silk-basedmaterials with properties comparable to native fibers. These mechanismssuggest new directions to design silk-based multifunctional materials. [ABSTRACT FROM AUTHOR]

Silk fibroin (SF) nanofibers, formed through electrospinning,haveattractive utility in regenerative medicine due to the biocompatibility,mechanical properties, and tailorable degradability. The mechanismof SF electrospun nanofiber formation was studied to gain new insightinto the formation and control of nanofibers. SF electrospinning solutionswith different nanostructures (nanospheres or nanofilaments) wereprepared by controlling the drying process during the preparationof regenerated SF films. Compared to SF nanospheres in solution, SFnanofilaments had better spinnability with lower viscosity when theconcentration of silk protein was below 10%, indicating a criticalrole for SF morphology, and in particular, nanostructures, for theformation of electrospun fibers. More interesting, the diameter ofelectrospun fibers gradually increased from 50 to 300 nm as the concentrationof SF nanofilaments in the solution increased from 6 to 12%, implyingsize control by simply adjusting SF nanostructure and concentration.Aside from process parameters investigated in previous studies, suchas SF concentration, viscosity, and electrical potential, the presentmechanism emphasizes significant influence of SF nanostructure onspinnability and diameter control of SF electrospun fibers, providinga controllable option for the preparation of silk-based electrospunscaffolds for biomaterials, drug delivery, and tissue engineeringneeds. [ABSTRACT FROM AUTHOR]

Novel protein chimeras constituted of “silk”anda silica-binding peptide (KSLSRHDHIHHH) were synthesized by geneticor chemical approaches and their influence on silica-silk based chimeracomposite formation evaluated. Genetic chimeras were constructed from6 or 15 repeats of the 32 amino acid consensus sequence of Nephila clavipesspider silk ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG]n) to which one silica binding peptide wasfused at the N terminus. For the chemical chimera, 28 equiv of thesilica binding peptide were chemically coupled to natural Bombyx morisilk after modification of tyrosine groups bydiazonium coupling and EDC/NHS activation of all acid groups. Aftersilica formation under mild, biomaterial-compatible conditions, theeffect of peptide addition on the properties of the silk and chimericsilk-silica composite materials was explored. The composite biomaterialproperties could be related to the extent of silica condensation andto the higher number of silica binding sites in the chemical chimeraas compared with the genetically derived variants. In all cases, thestructure of the protein/chimera in solution dictated the type ofcomposite structure that formed with the silica deposition processhaving little effect on the secondary structural composition of thesilk-based materials. Similarly to our study of genetic silk basedchimeras containing the R5 peptide (SSKKSGSYSGSKGSKRRIL), the roleof the chimeras (genetic and chemical) used in the present study residedmore in aggregation and scaffolding than in the catalysis of condensation.The variables of peptide identity, silk construct (number of consensusrepeats or silk source), and approach to synthesis (genetic or chemical)can be used to “tune” the properties of the compositematerials formed and is a general approach that can be used to preparea range of materials for biomedical and sensor-based applications. [ABSTRACT FROM AUTHOR]

The aim of the study was to determine the extent andmechanism of influence on silica condensation that is presented bya range of known silicifying recombinant chimeras (R5: SSKKSGSYSGSKGSKRRIL;A1: SGSKGSKRRIL; and Si4–1: MSPHPHPRHHHT and repeats thereof)attached at the N-terminus end of a 15-mer repeat of the 32 aminoacid consensus sequence of the major ampullate dragline Spindroin1 (Masp1) Nephila clavipesspider silk sequence ([SGRGGLGGQGAGAAAAAGGA GQGGYGGLGSQG]15X). The influence of the silk/chimeraratio was explored through the adjustment of the type and number ofsilicifying domains (denoted X above), and the results were comparedwith their non-chimeric counterparts and the silk from Bombyxmori. The effect of pH (3–9) on reactivity was alsoexplored. Optimum conditions for rate and control of silica depositionwere determined, and the solution properties of the silks were exploredto determine their mode(s) of action. For the silica–silk–chimeramaterials formed there is a relationship between the solution propertiesof the chimeric proteins (ability to carry charge), the pH of reaction,and the solid state materials that are generated. The region of colloidalinstability correlates with the pH range observed for morphologicalcontrol and coincides with the pH range for the highest silica condensationrates. With this information it should be possible to predict howchimeric or chemically modified proteins will affect structure andmorphology of materials produced under controlled conditions and extendthe range of composite materials for a wide spectrum of uses in thebiomedical and technology fields. [ABSTRACT FROM AUTHOR]

We demonstrate the assembly of extremely robust and pH-responsive thin shell LbL microcapsules from silk fibroin counterparts modified with poly-(lysine) and poly-(glutamic) acid, which are based on biocompatible silk ionomer materials in contrast with usually exploited synthetic polyelectrolytes. The microcapsules are extremely stable in an unusually wide pH range from 1.5 to 12.0 and show a remarkable degree of reversible swelling/deswelling response in dimensions, as exposed to extreme acidic and basic conditions. These changes are accompanied by reversible variations in shell permeability that can be utilized for pH-controlled loading and unloading of large macromolecules. Finally, we confirmed that these shells can be utilized to encapsulate yeast cells with a viability rate much higher than that for traditional synthetic polyelectrolytes. [ABSTRACT FROM AUTHOR]

We report on an application of silk as an ultrathin redox-active template for controllable, one-step synthesis of gold nanoparticles via control over silk secondary structure. We found that both silk I and silk II molecular layers can facilitate gold nanoparticle formation at ambient conditions, indicating that tyrosine groups are available for metal ion reduction in both forms of silk. We suggest that the presence of β-sheets in silk II facilitates tyrosine ordering thereby resulting in well-dispersed, uniform nanoparticles with diameters of less than 6 nm. In addition, the mineralization does not result in transformation of the silk I secondary structure to silk II. In fact, the silk I structure is stabilized from further transformation into silk II even upon drying. These results are critical for developing a better understanding of silk interfacial behavior and offer an opportunity to design a new class of nanocomposites that combine the beneficial features of silk with those of the nanoparticles. [ABSTRACT FROM AUTHOR]

Nanoscale surface patterning and polymerization of caffeic acid on 4-aminothiophenol-functionalized gold surfaces has been demonstrated with dip pen nanolithography (DPN). The diphenolic moiety of caffeic acid can be polymerized by biocatalysis with laccase or horseradish peroxidase. In the present study, the DPN patterned features were polymerized in situ through the use of the peroxidase. Using samples prepared by DPN, microcontact printing, and adsorption on macroscopic substrates, the products were characterized by electrostatic force microscopy (EFM), MALDI-TOF, X-ray photoelectron spectroscopy (XPS), UV-vis, and FT-IR. The in situ surface polymerization resulted in the formation of a quinone structure, while the phenyl ester formed in bulk polymerization reactions was not detected. A different coupling site was observed when comparing the polymers obtained from solution (bulk) vs the surface DPN reactions. The structural differences were attributed to surface-induced preorganization and orientation of the monomers prior to the enzymatic polymerization step. The results of this study expand the application of DPN technology to surface modification and surface chemistry reactions wherein stereoregularity and regioselectivity can be exploited. [ABSTRACT FROM AUTHOR]

Discusses the one-pot biocatalytic synthesis of novel amphiphilic products consisting of an ethyl glucopyranoside (EGP) headgroup and a hydrophobic chain. Materials and methods used; Information on work showing that ethylglucoside can be regioselectively monoacylagted with fatty acids by Candida antarctica lipase; Results and discussion pertaining to the study.