Phosphate anions-induced liquid-liquid phase separation in silk fibroin systems to produce biologically stable...

Silk fibroin (SF), the primary protein component of Bombyx mori silkworm cocoons, undergoes liquid-liquid phase separation (LLPS), followed by coacervation into fibers, in the silkworm glands. The molecular mechanisms underlying LLPS remain to be revealed. Here, we show that phosphate buffer (PB), a less commonly utilized, but more biomimetic route towards triggering SF assembly, induces LLPS of water-soluble SF by increasing hydrophobic interactions between SF chains. We demonstrate the ability of phosphate anions to promote self-assembly of silk fibroin through LLPS, resulting in protein-rich droplets. Complementary computational modeling using a bead-spring representation of SF supports the experimental findings and confirms the mechanistic origin of the assembly transitions, as driven primarily by hydrophobic interactions. FTIR spectroscopy was used to investigate structural differences upon LLPS between the dense and light phases, which were shown to be comprised mainly of random coil. After evaporation of the solvent, SF agglomerates were incorporated within the continuous silk matrix. This spatial confinement of solid droplets was stabilized by treatment with ethanol solution, promoting β-sheet formation via protein backbone dehydration. The material formulation was, finally, tested in simulated biological fluids (e.g., gastro-intestinal tract, based on European Pharmacopeia 9.0), highlighting the pH-dependent swelling, and overall stability of the films.