(149e) Engineering Charged Silk Conjugates for Ionically Crosslinked Hydrogels

Silk fibroin extracted from the fibers of the Bombyx mori silkworm’s cocoon is a high molecular weight protein whose versatility has encouraged its application to address a wide variety of biomedical challenges. B. mori fibroin consists of hydrophilic regions (blocks) that can be modified using a variety of aqueous and organic phase chemistries, as well as repeating hydrophobic regions (blocks) that associate by hydrogen bonding. This hydrogen bonding serves to physically cross-link the polymer chains, results in the formation of beta sheet secondary structures, and provides unique opportunity to tune the mechanical and degradation properties of the protein. In addition to its tunable properties, silk fibroin is biocompatible in a variety of biomedical applications in formats that include films, scaffolds, and hydrogels. The existing silk hydrogels are often formed by covalent cross-linking through dityrosine bond formation or by induction of the beta sheets, neither of which are reversible under cell compatible conditions. The goal of this work is to synthesize electrostatically crosslinked hydrogels using charged silk conjugates and to determine the extent to which the cross-linking process is reversible. This work utilizes an efficient synthetic route to carboxylate silk fibroin, a reaction that generates negatively charged silk conjugate materials. The reaction employed succinic anhydride in the presence of urea to modify the hydroxyl groups of the serine and threonine residues and amine groups of the lysine, arginine, and histidine residues, resulting in the enrichment of carboxylic acid functional groups. The addition of urea during the reaction led to a two-fold increase in the degree of protein carboxylation compared to existing methods, which is attributed to the disruption of hydrogen bonding in the hydrophobic sections of the protein. The increase in charged groups enabled the preparation of hydrogels via electrostatic interactions with oppositely charged molecules. Amine containing molecules (Jeffamine D and ED series) with varying molecular weights were mixed with carboxylated silk in a 1:1 molar charge ratio, which resulted in the formation of hydrogels via electrostatic crosslinking. The gelation kinetics were very slow for 1 w/v% carboxylated silk solution but were found to increase with increasing silk concentration up to 8 w/v%. Because the electrostatic interactions are non-covalent, this mode of crosslinking also offers the opportunity to prepare silk-based hydrogels that are reversible. The reversibility of gels was investigated by adding excess amine solution to hydrogels. The shear thinning behavior of the electrostatically crosslinked hydrogels was quantified by using dynamic shear rheology. The viability of human mesenchymal stem cells (hMSCs) encapsulated within the hydrogel was >90% as determined by Nucblue live reagent staining under fluorescence microscopy. The high viability indicated that the electrostatic network formed between carboxylated silk and Jeffamine ED showed no toxicity toward cells. Our ongoing work focuses on investigating reversibility through rheology and measuring degradation behavior of the silk hydrogels. Tuning the electrostatic charges on silk fibroin thus provides a viable route to synthesize electrostatically cross-linked hydrogels suitable for encapsulating hMSCs for soft tissue engineering applications.