Comprehensive Physicochemical Characterization and in Vitro Human Cell Culture Studies of an Innovative Biocompatible and Biodegradable Silk-Derived Protein Hydrolysate, SDP-4

SDP-4 is a soluble silk fibroin-derived protein hydrolysate extracted from the <i>Bombyx mori</i> silkworm cocoon and is a novel first-in-class biopolymer that is biodegradable, biocompatible, and shown to have regenerative properties. SDP-4 is currently used as a commercial wetting agent in topical eye drops, but it has also been shown to have anti-inflammatory properties that could be utilized in other biomedical applications. The purpose of this study was to comprehensively characterize the physicochemical properties that are necessary to design formulations and examine cell viability in response to varying doses of SDP-4 on different human cell types, with a particular attention toward respiratory applications. Lyophilized SDP-4 powder was characterized by scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, differential scanning calorimetry (DSC), hot-stage microscopy (HSM), Karl Fisher (KF) coulometric titration, Raman spectrometry, confocal Raman microscopy (CRM), and Fourier transform infrared microscopy. The lyophilized powder exhibited a nonuniform, angular glassy flake morphology with uniform chemical composition and minimal moisture uptake when tested under varying humidity conditions. Crystalline character was evident through birefringence at ambient temperature which changed during phase transitions, as evidenced through qualitative and quantitative assessments. Dose ranging SDP-4 biocompatibility studies on different human lung cells, nasal cells, skin cells, and brain cells was assessed by the in vitro cell viability assay. Assay results showed that cell viability was maintained at the various doses studied for different human cell types. The transepithelial resistance (TEER) assay showed that SDP-4 leads to transient fluctuations in cell membrane integrity and barrier tightness, followed by a recovery phase as cells adapt or repair the junctions. These findings demonstrate that SDP-4 is biocompatible with different types of human cells and safe at all of the doses studied. The unique physicochemical properties of SDP-4 revealed in this study demonstrate its favorable formulating ability for a variety of potential therapeutic applications.