Controlled radical polymerization of hydrophilic and zwitterionic brush-like polymers from silk fibroin surfaces.

Bombyx mori silk fibroin is a fibrous protein whose tunable properties and biocompatibility have resulted in its utility in a wide-variety of applications, including as drug delivery vehicles, wound dressings, and tissue engineering scaffolds. Control of protein and cell attachment is vital to the performance of biomaterials, but silk fibroin is mostly hydrophobic and interacts nonspecifically with cells and proteins. Silk functionalised with hydrophilic polymers reduces attachment, but the low number of reactive sites makes achieving a uniform conjugation a persistent challenge. This work presents a new approach to grow brush-like polymers from the surface of degradable silk films, where the films were enriched with hydroxyl groups, functionalised with an initiator, and finally reacted with acrylate monomers using atom transfer radical polymerisation. Two different routes to hydroxyl enrichment were investigated, one involving reaction with ethylene oxide (EO) and the other using a two-step photo-catalysed oxidation reaction. Both routes increased surface hydrophilicity, and hydrophilic monomers containing either uncharged (poly(ethylene glycol), PEG) pendant groups or zwitterionic pendant groups were polymerised from the surfaces. The initial processing of the films to induce beta sheet structures was found to impact the success of the polymerizations. Compared to the EO modified or unmodified silk surfaces, the oxidation reaction resulted in more polymer conjugation and the surfaces appear more uniform. Mesenchymal stem cell and protein attachment were the lowest on polymers grown from oxidised surfaces. PEG-containing brush-like polymers displayed lower protein attachment than surfaces conjugated with PEG using a previously reported "grafting to" method, but polymers containing zwitterionic side chains displayed both the lowest contact angles and the lowest cell and protein attachment. This finding may arise from the interactions of the zwitterionic pendant groups through their permanent dipoles and is an important finding because PEG is susceptible to oxidative damage that can reduce efficacy over time. These modified silk materials with lower cell and protein attachments are envisioned to find utility when enhanced diffusion around surfaces is required, such as in drug delivery implants.

Silk fibroin reactive inks for 3D printing crypt-like structures.

A reactive silk fibroin ink formulation designed for extrusion three-dimensional (3D) printing of protein-based hydrogels at room temperature is reported. This work is motivated by the need to produce protein hydrogels that can be printed into complex shapes with long-term stability using extrusion 3D printing at ambient temperature without the need for the addition of nanocomposites, synthetic polymers, or sacrifical templates. Silk fibroin from the Bombyx mori silkworm was purified and synthesized into reactive inks by enzyme-catalyzed dityrosine bond formation. Rheological and printing studies showed that tailoring the peroxide concentration in the reactive ink enables the silk to be extruded as a filament and printed into hydrogel constructs, supporting successive printed layers without flow of the construct or loss of desired geometry. To enable success of longer-term in vitro studies, 3D printed silk hydrogels were found to display excellent shape retention over time, as evidenced by no change in construct dimensions or topography when maintained for nine weeks in culture medium. Caco-2 (an intestinal epithelial cell line) attachment, proliferation, and tight junction formation on the printed constructs was not found to be affected by the geometry of the constructs tested. Intestinal myofibroblasts encapsulated within reactive silk inks were found to survive shearing during printing and proliferate within the hydrogel constructs. The work here thus provides a suitable route for extrusion 3D printing of protein hydrogel constructs that maintain their shape during printing and culture, and is expected to enable longer-term cellular studies of hydrogel constructs that require complex geometries and/or varying spatial distributions of cells on demand via digital printing.

Enhancing the Carboxylation Efficiency of Silk Fibroin through the Disruption of Noncovalent Interactions.

Silk fibroin is a semicrystalline protein used as a renewable polymer source and as a biomaterial platform, but existing methods to synthetically modify fibroin suffer from low efficiencies that can limit the protein's utility. This work reports on a mild synthesis that results in a 2-fold increase in carboxylation through the disruption of noncovalent interactions during the reaction. Importantly, silk fibroin maintains its ability to form ß-sheets that are critical for tailoring mechanical and degradation properties, as well as for rendering solid constructs (e.g., films and scaffolds) insoluble in water. Increasing carboxyl functionalization affords control over protein charge, which permits tailoring the loading and release of small molecules using electrostatic interactions. Disruption of noncovalent interactions during aqueous carbodiimide coupling also significantly enhances conjugation efficiency of molecules containing primary amine groups, thus enabling high degrees of functionalization with biological molecules, such as proteins and peptides, for biomaterial applications.

Dual-Mode Cross-Linking Enhances Adhesion of Silk Fibroin Hydrogels to Intestinal Tissue.

Compared to conventional wound closure methods like sutures and staples, polymer-based tissue adhesives afford some distinct advantages, such as greater ease of deployment in spatially constrained surgical sites. One way to achieve aqueous adhesion is by introducing catechol functional groups that form coordinate and covalent bonds with a variety of substrates. This approach, inspired by marine organisms, has been applied to biopolymers and synthetic polymers, but one key challenge is that compositions that are soluble in water are often susceptible to high swelling ratios that can result in undesired compression of neighboring tissues. This work sought to synthesize aqueous adhesive gels that are capable of two modes of association: (1) adhesion and covalent cross-linking reactions arising from catechol oxidation and (2) noncovalent cross-linking arising from self-assembly of polymer backbones within the gelled adhesive. The network's self-assembly after gelation was envisioned to afford control over swelling and reinforce its strength. Bombyx mori silk fibroin was selected as the backbone of the adhesive network because it can be processed into an aqueous solution yet later be rendered insoluble in water through the assembly of its hydrophobic protein core. Distinct from a previous approach to functionalize silk directly with catechol groups, this work investigated in situ generation of catechol on silk fibroin by enzymatically modifying phenolic side chains, where it was found that this enzymatic approach led to conjugates with higher degrees of catechol functionalization and aqueous solubility. Silk fibroin was functionalized with tyramine to enrich the protein's phenolic side chains, which were subsequently oxidized into catechol groups using mushroom tyrosinase (MT). The gelation of the silk conjugates with MT was monitored by rheology, and the gels exhibited low water uptake. Phenolic enrichment increased the rate of chemical cross-linking leading to gelation but did not interrupt assembly of silk's secondary structures. Adhesion of the tyramine-silk conjugates to porcine intestine was found to be superior to fibrin sealant, and induction of ß sheet secondary structures was found to further enhance adhesive strength through a second mode of cross-linking. Neither the chemical functionalization nor phenol oxidation affected the ability of intestinal epithelial cells (Caco-2) to attach and proliferate. Phenolic functionalization and oxidative cross-linking of silk fibroin was found to afford a new route to water-soluble, catechol-functionalized polymers, which were found to display excellent adhesion to mucosal tissue and whose secondary structure provides an additional mode to control strength and swelling of adhesive gels.