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.

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.

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.

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.

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.

Mechanical Enhancement of Bioinspired Polydopamine Nanocoatings.

Inspired by the catechol and amine-rich adhesive proteins of mussels, polydopamine (pDA) has become one of the most widely employed methods for functionalizing material surfaces, powered in part by the versatility and simplicity of pDA film deposition that takes place spontaneously on objects immersed in an alkaline aqueous solution of dopamine monomer. Despite the widespread adoption of pDA as a multifunctional coating for surface modification, it exhibits poor mechanical performance. Attempts to modify the physical properties of pDA by incorporation of oxidizing agents, cross-linkers, or carbonization of the films at ultrahigh temperatures have been reported; however, improving mechanical properties with mild post-treatments without sacrificing the functionality and versatility of pDA remains a challenge. Here, we demonstrate thermal annealing at a moderate temperature (130 °C) as a facile route to enhance mechanical robustness of pDA coatings. Chemical spectroscopy, X-ray scattering, molecular force spectroscopy, and bulk mechanical analyses indicate that monomeric and oligomeric species undergo further polymerization during thermal annealing, leading to fundamental changes in molecular and bulk mechanical behavior of pDA. Considerable improvements in scratch resistance were noted in terms of both penetration depth (32% decrease) and residual depth (74% decrease) for the annealed pDA coating, indicating the enhanced ability of the annealed coating to resist mechanical deformations. Thermal annealing resulted in significant enhancement in the intermolecular and cohesive interactions between the chains in the pDA structure, attributed to cross-linking and increased entanglements, preventing desorption and detachment of the chains from the coating. Importantly, improvements in pDA mechanical performance through thermal annealing did not compromise the ability of pDA to support secondary coating reactions as evidenced by electroless deposition of a metal film adlayer on annealed pDA.

Universal nanothin silk coatings via controlled spidroin self-assembly.

Robust, biocompatible, and facile coatings are promising for improving the in vivo performance of medical implants and devices. Here, we demonstrate the formation of nanothin silk coatings by leveraging the biomimetic self-assembly of eADF4(C16), an amphiphilic recombinant protein based on the Araneus diadematus dragline spidroin ADF4. These coatings result from concurrent adsorption and supramolecular assembly of eADF4(C16) induced by KH2PO4, thereby providing a mild one-pot coating strategy in which the coating rate can be controlled by protein and KH2PO4 concentration. The thickness of the coatings ranges from 2-30 nm depending on the time immersed in the aqueous coating solution. Coatings can be formed on hydrophobic and hydrophilic substrates regardless of surface chemistry and without requiring specialized surface activation. Moreover, coatings appear to be stable through vigorous rinsing and prolonged agitation in water. Grazing incidence wide angle X-ray scattering, single-molecule force spectroscopy, and Congo red staining techniques confirm the formation of ß-sheet nanocrystals within the eADF4(C16) coating, which contributes to the cohesive and adhesive stability of the material. Coatings are exceptionally smooth in the dry state and are hydrophilic regardless of substrate hydrophobicity. Under aqueous conditions, nanothin silk coatings exhibit the properties of a hydrogel material.