Mechanisms of Silk-Derived Protein (SDP) Hydrolysate Stability within Aqueous Formulation and In Vitro Assessment of NF-kB Inflammatory Pathway Inhibition

Abstract

Background Silk fibroin is a structural protein that can be regenerated into aqueous solution, and then used for a variety of biomedical and advanced material applications due to its high biocompatibility and controllable material properties. Conversely, fibroin solution can have limited utility due to its inherent physical instability to self-associate into higher order structures. Here we describe a fibroin hydrolysate, termed silk-derived protein (SDP), which mimics the same manufacturing process as aqueous silk fibroin but introduces an additional hydrolysis step. Methods The biochemical properties and material stability mechanisms of SDP were characterized through various assessments, including MWD, amino acid content, solubility measurements, surface interaction, and protein secondary structure formation. Additional in vitro studies were undertaken to assess SDP’s ability to inhibit NF-kB-mediated inflammation and mRNA transcription. Results SDP was found to have enhanced solubility, stability, and surface wetting properties when added to aqueous formulation reaching over 40% wt./vol. concentration and a viscosity of 140 mPa. Mechanistic stability studies indicate that the combination of heating, pressure and LiBr is required to enhance hydrolysate stability by abolishing fibroin’s ability to self-associate through the formation of β-sheet secondary structures. In vitro assays using the HCLE cell lines indicated SDP had dose dependent potency for inhibiting translocation of the p65 transcription factor into the nucleus with, while showing multi-fold reduction in NF-kB driven TNF-α and MMP-9 gene expression. Conclusions Collectively, the results support SDP’s use as an anti-inflammatory wetting agent compatible with a wide range of both biomedical and industrial applications, and offers a sustainable biomaterial alternative to existing anti-inflammatories, surfactants, and demulcents that possess higher toxicity profiles.