Enhanced Hydrogen Bonding by Urea Functionalization Tunes the Stability and Biological Properties of Peptide Amphiphiles.

Self-assembled nanostructures such as those formed by peptide amphiphiles (PAs) are of great interest in biological and pharmacological applications. Herein, a simple and widely applicable chemical modification, a urea motif, was included in the PA's molecular structure to stabilize the nanostructures by virtue of intermolecular hydrogen bonds. Since the amino acid residue nearest to the lipid tail is the most relevant for stability, we decided to include the urea modification at that position. We prepared four groups of molecules (13 PAs in all), with varying levels of intermolecular cohesion, using amino acids with distinct ß-sheet promoting potential and/or containing hydrophobic tails of distinct lengths. Each subset contained one urea-modified PA and nonmodified PAs, all with the same peptide sequence. The varied responses of these PAs to variations in pH, temperature, counterions, and biologically related proteins were examined using microscopic, X-ray, spectrometric techniques, and molecular simulations. We found that the urea group contributes to the stabilization of the morphology and internal arrangement of the assemblies against environmental stimuli for all peptide sequences. In addition, microbiological and biological studies were performed with the cationic PAs. These assays reveal that the addition of urea linkages affects the PA-cell membrane interaction, showing the potential to increase the selectivity toward bacteria. Our data indicate that the urea motif can be used to tune the stability of a wide range of PA nanostructures, allowing flexibility on the biomaterial's design and opening a myriad of options for clinical therapies.

Production and secretion of recombinant spider silk in Bacillus megaterium.

BACKGROUND: Silk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider silk, known for its exceptional mechanical strength, has attracted considerable attention. Recombinant production of spider silk represents the most promising route towards its scaled production; however, challenges persist within the upstream optimization of host organisms, including toxicity and low yields. The high cost of downstream cell lysis and protein purification is an additional barrier preventing the widespread production and use of spider silk proteins. Gram-positive bacteria represent an attractive, but underexplored, microbial chassis that may enable a reduction in the cost and difficulty of recombinant silk production through attributes that include, superior secretory capabilities, frequent GRAS status, and previously established use in industry. RESULTS: In this study, we explore the potential of gram-positive hosts by engineering the first production and secretion of recombinant spider silk in the Bacillus genus. Using an industrially relevant B. megaterium host, it was found that the Sec secretion pathway enables secretory production of silk, however, the choice of signal sequence plays a vital role in successful secretion. Attempts at increasing secreted titers revealed that multiple translation initiation sites in tandem do not significantly impact silk production levels, contrary to previous findings for other gram-positive hosts and recombinant proteins. Notwithstanding, targeted amino acid supplementation in minimal media was found to increase production by 135% relative to both rich media and unaltered minimal media, yielding secretory titers of approximately 100 mg/L in flask cultures. CONCLUSION: It is hypothesized that the supplementation strategy addressed metabolic bottlenecks, specifically depletion of ATP and NADPH within the central metabolism, that were previously observed for an E. coli host producing the same recombinant silk construct. Furthermore, this study supports the hypothesis that secretion mitigates the toxicity of the produced silk protein on the host organism and enhances host performance in glucose-based minimal media. While promising, future research is warranted to understand metabolic changes more precisely in the Bacillus host system in response to silk production, optimize signal sequences and promoter strengths, investigate the mechanisms behind the effect of tandem translation initiation sites, and evaluate the performance of this system within a bioreactor.

Potential enthalpic energy of water in oils exploited to control supramolecular structure.

Water directs the self-assembly of both natural1,2 and synthetic3-9 molecules to form precise yet dynamic structures. Nevertheless, our molecular understanding of the role of water in such systems is incomplete, which represents a fundamental constraint in the development of supramolecular materials for use in biomaterials, nanoelectronics and catalysis 10 . In particular, despite the widespread use of alkanes as solvents in supramolecular chemistry11,12, the role of water in the formation of aggregates in oils is not clear, probably because water is only sparingly miscible in these solvents-typical alkanes contain less than 0.01 per cent water by weight at room temperature 13 . A notable and unused feature of this water is that it is essentially monomeric 14 . It has been determined previously 15 that the free energy cost of forming a cavity in alkanes that is large enough for a water molecule is only just compensated by its interaction with the interior of the cavity; this cost is therefore too high to accommodate clusters of water. As such, water molecules in alkanes possess potential enthalpic energy in the form of unrealized hydrogen bonds. Here we report that this energy is a thermodynamic driving force for water molecules to interact with co-dissolved hydrogen-bond-based aggregates in oils. By using a combination of spectroscopic, calorimetric, light-scattering and theoretical techniques, we demonstrate that this interaction can be exploited to modulate the structure of one-dimensional supramolecular polymers.

Silk fibroin production in Escherichia coli is limited by a positive feedback loop between metabolic burden and toxicity stress.

To investigate the metabolic elasticity and production bottlenecks for recombinant silk proteins in Escherichia coli, we performed a comprehensive characterization of one elastin-like peptide strain (ELP) and two silk protein strains (A5 4mer, A5 16mer). Our approach included 13C metabolic flux analysis, genome-scale modeling, transcription analysis, and 13C-assisted media optimization experiments. Three engineered strains maintained their central flux network during growth, while measurable metabolic flux redistributions (such as the Entner-Doudoroff pathway) were detected. Under metabolic burdens, the reduced TCA fluxes forced the engineered strain to rely more on substrate-level phosphorylation for ATP production, which increased acetate overflow. Acetate (as low as 10 mM) in the media was highly toxic to silk-producing strains, which reduced 4mer production by 43% and 16mer by 84%, respectively. Due to the high toxicity of large-size silk proteins, 16mer's productivity was limited, particularly in the minimal medium. Therefore, metabolic burden, overflow acetate, and toxicity of silk proteins may form a vicious positive feedback loop that fractures the metabolic network. Three solutions could be applied: 1) addition of building block supplements (i.e., eight key amino acids: His, Ile, Phe, Pro, Tyr, Lys, Met, Glu) to reduce metabolic burden; 2) disengagement of growth and production; and 3) use of non-glucose based substrate to reduce acetate overflow. Other reported strategies were also discussed in light of decoupling this positive feedback loop.

Two-step conversion of polyethylene into recombinant proteins using a microbial platform.

BACKGROUND: The increasing prevalence of plastic waste combined with the inefficiencies of mechanical recycling has inspired interest in processes that can convert these waste streams into value-added biomaterials. To date, the microbial conversion of plastic substrates into biomaterials has been predominantly limited to polyhydroxyalkanoates production. Expanding the capabilities of these microbial conversion platforms to include a greater diversity of products generated from plastic waste streams can serve to promote the adoption of these technologies at a larger scale and encourage a more sustainable materials economy. RESULTS: Herein, we report the development of a new strain of Pseudomonas bacteria capable of converting depolymerized polyethylene into high value bespoke recombinant protein products. Using hexadecane, a proxy for depolymerized polyethylene, as a sole carbon nutrient source, we optimized media compositions that facilitate robust biomass growth above 1 × 109 cfu/ml, with results suggesting the benefits of lower hydrocarbon concentrations and the use of NH4Cl as a nitrogen source. We genomically integrated recombinant genes for green fluorescent protein and spider dragline-inspired silk protein, and we showed their expression in Pseudomonas aeruginosa, reaching titers of approximately 10 mg/L when hexadecane was used as the sole carbon source. Lastly, we demonstrated that chemically depolymerized polyethylene, comprised of a mixture of branched and unbranched alkanes, could be converted into silk protein by Pseudomonas aeruginosa at titers of 11.3 ± 1.1 mg/L. CONCLUSION: This work demonstrates a microbial platform for the conversion of a both alkanes and plastic-derived substrates to recombinant, protein-based materials. The findings in this work can serve as a basis for future endeavors seeking to upcycle recalcitrant plastic wastes into value-added recombinant proteins.

Rapid Synthesis of Silk-Like Polymers Facilitated by Microwave Irradiation and Click Chemistry.

Silk is a natural fiber that surpasses most man-made polymers in its combination of strength and toughness. Silk fibroin, the primary protein component of silk, can be synthetically mimicked by a linear copolymer with alternating rigid and soft segments. Strategies for chemical synthesis of such silk-like polymers have persistently resulted in poor sequence control, long reaction times, and low molecular weights. Here, we present a two-stage approach for rapidly synthesizing silk-like polymers with precisely defined rigid blocks. This approach utilizes solid-phase peptide synthesis to create uniform oligoalanine "prepolymers", followed by microwave-assisted step-growth polymerization with bifunctional poly(ethylene glycol). Multiple coupling chemistries and reaction conditions were explored, with microwave-assisted click chemistry yielding polymers with Mw ∼ 14 kg/mol in less than 20 min. These polymers formed antiparallel ß-sheets and nanofibers, which is consistent with the structure of natural silk fibroin. Thus, our strategy demonstrates a promising modular approach for synthesizing silk-like polymers.

Self-Assembly-Assisted Kinetically Controlled Papain-Catalyzed Formation of mPEG-b-Phe(Leu)x.

Self-assembling peptide materials are promising next-generation materials with applications that include tissue engineering scaffolds, drug delivery, bionanomedicine, and enviro-responsive materials. Despite these advances, synthetic methods to form peptides and peptide-polymer conjugates still largely rely on solid-phase peptide synthesis (SPPS) and N-carboxyanhydride ring-opening polymerization (NCA-ROP), while green methods remain largely undeveloped. This work demonstrates a protease-catalyzed peptide synthesis (PCPS) capable of directly grafting leucine ethyl ester (Leu-OEt) from the C-terminus of a methoxy poly(ethylene glycol)-phenylalanine ethyl ester macroinitiator in a one-pot, aqueous reaction. By using the natural tendency of the growing hydrophobic peptide segment to self-assemble, a large narrowing of the (Leu)x distributions for both mPEG45-b-Phe(Leu)x and oligo(Leu)x coproducts, relative to oligo(Leu)x synthesized in the absence of a macroinitiator (mPEG45-Phe-OEt), was achieved. A mechanism is described where in situ ß-sheet coassembly of mPEG45-b-Phe(Leu)x and oligo(Leu)x coproducts during polymerization prevents peptide hydrolysis, providing a means to control the degree of polymerization (DP) and dispersity of diblock (Leu)x segments (matrix-assisted laser desorption time-of-flight (MALDI-TOF) x = 5.1, dispersity ≤ 1.02). The use of self-assembly to control the uniformity of peptides synthesized by PCPS paves the way for precise peptide block copolymer architectures with various polymer backbones and amino acid compositions synthesized by a green process.

Silk and Silk-Like Supramolecular Materials.

Silk is a source of marvel for centuries as one of nature's high-performance materials. More recently, chemical and structural analysis techniques have helped explore the relationship between silk's properties and its hierarchical structure. Furthermore, recombinant protein engineering as well as polymer and organic synthesis techniques have enabled the production of silk-like materials. It has become apparent that silk is a supramolecular polymer with many of the properties exhibited by well-known synthetic supramolecular materials, such as block copolymers, liquid crystals, thermoplastic elastomers, and self-assembling peptides. In this review, the hierarchical structure and supramolecular assembly of silk are discussed in comparison to these synthetic supramolecular systems. By focusing on the connections between chemical structure, nanoscale molecular organization, and material properties, the aim is to provide perspectives on the rational design of advanced soft matter to supramolecular chemists and molecular engineers who look to nature for inspiration.

Pathway Complexity in the Enantioselective Self-Assembly of Functional Carbonyl-Bridged Triarylamine Trisamides.

Functional supramolecular systems like carbonyl-bridged triarylamine (CBT) trisamides are known for their long-range energy transport at room temperature. Understanding the complex self-assembly processes of this system allows for control over generated structures using controlled supramolecular polymerization. Here, we present two novel CBT trisamides with (S)- or (R)-chiral side chains which show a two-pathway self-assembly behavior in solution. Depending on the thermal profile during the self-assembly process, two different stable states are obtained under otherwise identical conditions. A kinetically trapped state A is reached upon cooling to 7 °C, via a proposed isodesmic process. In addition, there is a thermodynamically stable state B at 7 °C that is induced by first undercooling to -5 °C, via a nucleation-elongation mechanism. In both cases, helical supramolecular aggregates comprising H-aggregated CBTs are formed. Additionally, controlled supramolecular polymerization was achieved by mixing the two different states (A and B) from the same enantiomer, leading to a conversion of the kinetically trapped state to the thermodynamically stable state. This process is highly enantioselective, as no conversion is observed if the two states consist of opposite enantiomers. We thus show the importance and opportunities emerging from understanding the pathway complexity of functional supramolecular systems.

End Groups of Functionalized Siloxane Oligomers Direct Block-Copolymeric or Liquid-Crystalline Self-Assembly Behavior.

Monodisperse oligodimethylsiloxanes end-functionalized with the hydrogen-bonding ureidopyrimidinone (UPy) motif undergo phase separation between their aromatic end groups and dimethylsiloxane midblocks to form ordered nanostructures with domain spacings of <5 nm. The self-assembly behavior of these well-defined oligomers resembles that of high degree of polymerization (N)-high block interaction parameter (χ) linear diblock copolymers despite their small size. Specifically, the phase morphology varies from lamellar to hexagonal to body-centered cubic with increasing asymmetry in molecular volume fraction. Mixing molecules with different molecular weights to give dispersity >1.13 results in disorder, showing importance of molecular monodispersity for ultrasmall ordered phase separation. In contrast, oligodimethylsiloxanes end-functionalized with an O-benzylated UPy derivative self-assemble into lamellar nanostructures regardless of volume fraction because of the strong preference of the end groups to aggregate in a planar geometry. Thus, these molecules display more classically liquid-crystalline self-assembly behavior where the lamellar bilayer thickness is determined by the siloxane midblock. Here the lamellar nanostructure is tolerant to molecular polydispersity. We show the importance of end groups in high χ-low N block molecules, where block-copolymer-like self-assembly in our UPy-functionalized oligodimethylsiloxanes relies upon the dominance of phase separation effects over directional end group aggregation.

Molecular design for growth of supramolecular membranes with hierarchical structure.

Membranes with hierarchical structure exist in biological systems, and bio-inspired building blocks have been used to grow synthetic analogues in the laboratory through self-assembly. The formation of these synthetic membranes is initiated at the interface of two aqueous solutions, one containing cationic peptide amphiphiles (PA) and the other containing the anionic biopolymer hyaluronic acid (HA). The membrane growth process starts within milliseconds of interface formation and continues over much longer timescales to generate robust membranes with supramolecular PA-HA nanofibers oriented orthogonal to the interface. Computer simulation indicates that formation of these hierarchically structured membranes requires strong interactions between molecular components at early time points in order to generate a diffusion barrier between both solutions. Experimental studies using structurally designed PAs confirm simulation results by showing that only PAs with high ζ potential are able to yield hierarchically structured membranes. Furthermore, the chemical structure of such PAs must incorporate residues that form ß-sheets, which facilitates self-assembly of long nanofibers. In contrast, PAs that form low aspect ratio nanostructures interact weakly with HA and yield membranes that exhibit non-fibrous fingering protrusions. Furthermore, experimental results show that increasing HA molecular weight decreases the growth rate of orthogonal nanofibers. This result is supported by simulation results suggesting that the thickness of the interfacial contact layer generated immediately after initiation of self-assembly increases with polymer molecular weight.

Electrostatic control of structure in self-assembled membranes.

Self-assembling peptide amphiphiles (PAs) can form hierarchically ordered membranes when brought in contact with aqueous polyelectrolytes of the opposite charge by rapidly creating a diffusion barrier composed of filamentous nanostructures parallel to the plane of the incipient membrane. Following this event, osmotic forces and charge complexation template nanofiber growth perpendicular to the plane of the membrane in a dynamic self-assembly process. In this work, we show that this hierarchical structure requires massive interfacial aggregation of PA molecules, suggesting the importance of rapid diffusion barrier formation. Strong PA aggregation is induced here through the use of heparin-binding PAs with heparin and also with polyelectrolytes of varying charge density. Small angle X-ray scattering shows that in the case of weak PA-polyelectrolyte interaction, membranes formed display a cubic phase ordering on the nanoscale that likely results from clusters of PA nanostructures surrounded by polyelectrolyte chains.

One-Pot Assembly of Drug-Eluting Silk Coatings with Applications for Nerve Regeneration.

Clinical use of polymeric scaffolds for tissue engineering often suffers from their inability to promote strong cellular interactions. Functionalization with biomolecules may improve outcomes; however, current functionalization approaches using covalent chemistry or physical adsorption can lead to loss of biomolecule bioactivity. Here, we demonstrate a novel bottom-up approach for enhancing the bioactivity of poly(l-lactic acid) electrospun scaffolds though interfacial coassembly of protein payloads with silk fibroin into nanothin coatings. In our approach, protein payloads are first added into an aqueous solution with Bombyx mori-derived silk fibroin. Phosphate anions are then added to trigger coassembly of the payload and silk fibroin, as well as noncovalent formation of a payload-silk fibroin coating at poly(l-lactic) acid fiber surfaces. Importantly, the coassembly process results in homogeneous distribution of protein payloads, with the loading quantity depending on payload concentration in solution and coating time. This coassembly process yields greater loading capacity than physical adsorption methods, and the payloads can be released over time in physiologically relevant conditions. We also demonstrate that the coating coassembly process can incorporate nerve growth factor and that coassembled coatings lead to significantly more neurite extension than loading via adsorption in a rat dorsal root ganglia explant culture model.

Dry and wet wrinkling of a silk fibroin biopolymer by a shape-memory material with insight into mechanical effects on secondary structures in the silk network.

Surface wrinkling provides an approach to modify the surfaces of biomedical devices to better mimic features of the extracellular matrix and guide cell attachment, proliferation, and differentiation. Biopolymer wrinkling on active materials holds promise but is poorly explored. Here we report a mechanically actuated assembly process to generate uniaxial micro-and nanosized silk fibroin (SF) wrinkles on a thermo-responsive shape-memory polymer (SMP) substrate, with wrinkling demonstrated under both dry and hydrated (cell compatible) conditions. By systematically investigating the influence of SMP programmed strain magnitude, film thickness, and aqueous media on wrinkle stability and morphology, we reveal how to control the wrinkle sizes on the micron and sub-micron length scale. Furthermore, as a parameter fundamental to SMPs, we demonstrate that the temperature during the recovery process can also affect the wrinkle characteristics and the secondary structures in the silk network. We find that with increasing SMP programmed strain magnitude, silk wrinkled topographies with increasing wavelengths and amplitudes are achieved. Furthermore, silk wrinkling is found to increase ß-sheet content, with spectroscopic analysis suggesting that the effect may be due primarily to tensile (e.g., Poisson effect and high-curvature wrinkle) loading modes in the SF, despite the compressive bulk deformation (uniaxial contraction) used to produce wrinkles. Silk wrinkles fabricated from sufficiently thick films (roughly 250 nm) persist after 24 h in cell culture medium. Using a fibroblast cell line, analysis of cellular response to the wrinkled topographies reveals high viability and attachment. These findings demonstrate use of wrinkled SF films under physiologically relevant conditions and suggest the potential for biopolymer wrinkles on biomaterials surfaces to find application in cell mechanobiology, wound healing, and tissue engineering.

Novel insights into construct toxicity, strain optimization, and primary sequence design for producing recombinant silk fibroin and elastin-like peptide in E. coli.

Spider silk proteins (spidroins) are a remarkable class of biomaterials that exhibit a unique combination of high-value attributes and can be processed into numerous morphologies for targeted applications in diverse fields. Recombinant production of spidroins represents the most promising route towards establishing the industrial production of the material, however, recombinant spider silk production suffers from fundamental difficulties that includes low titers, plasmid instability, and translational inefficiencies. In this work, we sought to gain a deeper understanding of upstream bottlenecks that exist in the field through the production of a panel of systematically varied spidroin sequences in multiple E. coli strains. A restriction on basal expression and specific genetic mutations related to stress responses were identified as primary factors that facilitated higher titers of the recombinant silk constructs. Using these findings, a novel strain of E. coli was created that produces recombinant silk constructs at levels 4-33 times higher than standard BL21(DE3). However, these findings did not extend to a similar recombinant protein, an elastin-like peptide. It was found that the recombinant silk proteins, but not the elastin-like peptide, exert toxicity on the E. coli host system, possibly through their high degree of intrinsic disorder. Along with strain engineering, a bioprocess design that utilizes longer culturing times and attenuated induction was found to raise recombinant silk titers by seven-fold and mitigate toxicity. Targeted alteration to the primary sequence of the recombinant silk constructs was also found to mitigate toxicity. These findings identify multiple points of focus for future work seeking to further optimize the recombinant production of silk proteins and is the first work to identify the intrinsic disorder and subsequent toxicity of certain spidroin constructs as a primary factor related to the difficulties of production.

Electrospun fiber-mediated delivery of neurotrophin-3 mRNA for neural tissue engineering applications.

Aligned electrospun fibers provide topographical cues and local therapeutic delivery to facilitate robust peripheral nerve regeneration. mRNA delivery enables transient expression of desired proteins that promote axonal regeneration. However, no prior work delivers mRNA from electrospun fibers for peripheral nerve regeneration applications. Here, we developed the first aligned electrospun fibers to deliver pseudouridine-modified (Ψ) neurotrophin-3 (NT-3) mRNA (ΨNT-3mRNA) to primary Schwann cells and assessed NT-3 secretion and bioactivity. We first electrospun aligned poly(L-lactic acid) (PLLA) fibers and coated them with the anionic substrates dextran sulfate sodium salt (DSS) or poly(3,4-dihydroxy-L-phenylalanine) (pDOPA). Cationic lipoplexes containing ΨNT-3mRNA complexed to JetMESSENGER® were then immobilized to the fibers, resulting in detectable ΨNT-3mRNA release for 28 days from all fiber groups investigated (PLLA+mRNA, 0.5DSS4h+mRNA, and 2pDOPA4h+mRNA). The 2pDOPA4h+mRNA group significantly increased Schwann cell secretion of NT-3 for 21 days compared to control PLLA fibers (p < 0.001-0.05) and, on average, increased Schwann cell secretion of NT-3 by ≥ 2-fold compared to bolus mRNA delivery from the 1µgBolus+mRNA and 3µgBolus+mRNA groups. The 2pDOPA4h+mRNA fibers supported Schwann cell secretion of NT-3 at levels that significantly increased dorsal root ganglia (DRG) neurite extension by 44% (p < 0.0001) and neurite area by 64% (p < 0.001) compared to control PLLA fibers. The data show that the 2pDOPA4h+mRNA fibers enhance the ability of Schwann cells to promote neurite growth from DRG, demonstrating this platform's potential capability to improve peripheral nerve regeneration. STATEMENT OF SIGNIFICANCE: Aligned electrospun fibers enhance axonal regeneration by providing structural support and guidance cues, but further therapeutic stimulation is necessary to improve functional outcomes. mRNA delivery enables the transient expression of therapeutic proteins, yet achieving local, sustained delivery remains challenging. Previous work shows that genetic material delivery from electrospun fibers improves regeneration; however, mRNA delivery has not been explored. Here, we examine mRNA delivery from aligned electrospun fibers to enhance neurite outgrowth. We show that immobilization of NT-3mRNA/JetMESSENGER® lipoplexes to aligned electrospun fibers functionalized with pDOPA enables local, sustained NT-3mRNA delivery to Schwann cells, increasing Schwann cell secretion of NT-3 and enhancing DRG neurite outgrowth. This study displays the potential benefits of electrospun fiber-mediated mRNA delivery platforms for neural tissue engineering.

Virucidal N95 Respirator Face Masks via Ultrathin Surface-Grafted Quaternary Ammonium Polymer Coatings.

N95 respirator face masks serve as effective physical barriers against airborne virus transmission, especially in a hospital setting. However, conventional filtration materials, such as nonwoven polypropylene fibers, have no inherent virucidal activity, and thus, the risk of surface contamination increases with wear time. The ability of face masks to protect against infection can be likely improved by incorporating components that deactivate viruses on contact. We present a facile method for covalently attaching antiviral quaternary ammonium polymers to the fiber surfaces of nonwoven polypropylene fabrics that are commonly used as filtration materials in N95 respirators via ultraviolet (UV)-initiated grafting of biocidal agents. Here, C12-quaternized benzophenone is simultaneously polymerized and grafted onto melt-blown or spunbond polypropylene fabric using 254 nm UV light. This grafting method generated ultrathin polymer coatings which imparted a permanent cationic charge without grossly changing fiber morphology or air resistance across the filter. For melt-blown polypropylene, which comprises the active filtration layer of N95 respirator masks, filtration efficiency was negatively impacted from 72.5 to 51.3% for uncoated and coated single-ply samples, respectively. Similarly, directly applying the antiviral polymer to full N95 masks decreased the filtration efficiency from 90.4 to 79.8%. This effect was due to the exposure of melt-blown polypropylene to organic solvents used in the coating process. However, N95-level filtration efficiency could be achieved by wearing coated spunbond polypropylene over an N95 mask or by fabricating N95 masks with coated spunbond as the exterior layer. Coated materials demonstrated broad-spectrum antimicrobial activity against several lipid-enveloped viruses, as well as Staphylococcus aureus and Escherichia coli bacteria. For example, a 4.3-log reduction in infectious MHV-A59 virus and a 3.3-log reduction in infectious SuHV-1 virus after contact with coated filters were observed, although the level of viral deactivation varied significantly depending on the virus strain and protocol for assaying infectivity.

Peptide Self-Assembly for Crafting Functional Biological Materials.

Self-assembling, peptide-based scaffolds are frontrunners in the search for biomaterials with widespread impact in regenerative medicine. The inherent biocompatibility and cell signaling capabilities of peptides, in combination with control of secondary structure, has led to the development of a broad range of functional materials with potential for many novel therapies. More recently, membranes formed through complexation of peptide nanostructures with natural biopolymers have led to the development of hierarchically-structured constructs with potentially far-reaching applications in biology and medicine. In this review, we highlight recent advances in peptide-based gels and membranes, including work from our group and others. Specifically, we discuss the application of peptide-based materials in the regeneration of bone and enamel, cartilage, and the central nervous system, as well as the transplantation of islets, wound-healing, cardiovascular therapies, and treatment of erectile dysfunction after prostatectomy.

Coating Topologically Complex Electrospun Fibers with Nanothin Silk Fibroin Enhances Neurite Outgrowth in Vitro.

Electrospun poly-l-lactic acid (PLLA) fibers are commonly used for tissue engineering applications because of their uniform morphology, and their efficacy can be further enhanced via surface modification. In this study, we aimed to increase neurite outgrowth along electrospun fibers by coating with silk fibroin (SF), a bioinert protein derived from Bombyx mori cocoon threads, shown to be neurocompatible. Aligned PLLA fibers were electrospun with smooth, pitted, and divoted surface nanotopographies and coated with SF by immersion in coating solution for either 12 or 24 h. Specifically, thin-film coatings of SF were generated by leveraging the controlled self-assembly of SF in aqueous conditions that promote ß-sheet assembly. For both 12- and 24-h coatings, Congo Red staining for ß-sheet structures confirmed the presence of SF coatings on PLLA fibers. Confocal imaging of fluorescein-labeled SF further demonstrated a homogeneous coating formation on PLLA fibers. No change in the water contact angle of the surfaces was observed after coating; however, an increase in the isoelectric point (pI) to values comparable with the theoretical pI of SF was seen. Notably, there was a significant trend of increased dorsal root ganglia (DRG) adhesion on scaffolds coated with SF, as well as greater neurite outgrowth on pitted and divoted fibers that had been coated with SF. Ultimately, this work demonstrated that thin-film SF coatings formed by self-assembly uniformly coat electrospun fibers, providing a new strategy to increase the neuroregenerative capacity of electrospun scaffolds. To our knowledge, this is the first instance of biomedical modification of topologically complex substrates using noncovalent methods.

Chemical Synthesis of Silk-Mimetic Polymers.

Silk is a naturally occurring high-performance material that can surpass man-made polymers in toughness and strength. The remarkable mechanical properties of silk result from the primary sequence of silk fibroin, which bears semblance to a linear segmented copolymer with alternating rigid ("crystalline") and flexible ("amorphous") blocks. Silk-mimetic polymers are therefore of great emerging interest, as they can potentially exhibit the advantageous features of natural silk while possessing synthetic flexibility as well as non-natural compositions. This review describes the relationships between primary sequence and material properties in natural silk fibroin and furthermore discusses chemical approaches towards the synthesis of silk-mimetic polymers. In particular, step-growth polymerization, controlled radical polymerization, and copolymerization with naturally derived silk fibroin are presented as strategies for synthesizing silk-mimetic polymers with varying molecular weights and degrees of sequence control. Strategies for improving macromolecular solubility during polymerization are also highlighted. Lastly, the relationships between synthetic approach, supramolecular structure, and bulk material properties are explored in this review, with the aim of providing an informative perspective on the challenges facing chemical synthesis of silk-mimetic polymers with desirable properties.