(113i) Continuous Nano-Thin Coating Formation By Silk Fibroin Self-Assembly

Silk fibroin is a fiber-forming protein derived from the thread of Bombyx mori silkworm cocoons. This biocompatible protein, under the influence of kosmotropic agents such as potassium phosphate, can undergo self-assembly to transform from a water-soluble protein to a robust β-sheet rich elastomeric material. By leveraging the concurrent non-specific adsorption and self-assembly of silk fibroin, we have demonstrated the non-covalent growth of adherent and defect-free nano-thin silk fibroin coatings on a variety of surfaces. These coatings can functionalize topographically complex surfaces without requiring chemical derivatization or specific adhesive interactions, showing potential as a versatile surface modification strategy for biomedical applications.

Here, we explore the fundamental mechanisms of coating growth by examining the effects of coating solution parameters which promote or inhibit silk fibroin self-assembly. These parameters include protein concentration, pH, time, and kosmotropic and chaotropic salts from the Hofmeister series, on the solution-phase assembly and interfacial accumulation of silk fibroin. Coating kinetics, measured using quartz crystal microbalance with dissipation, shows a strong dependence on pH, salt species, and salt concentration. In kosmotropic solution conditions that promote inter-protein interactions, such as low potassium phosphate concentrations (100 – 300 mM), silk fibroin coatings display continual growth over time with no apparent saturation, a behavior not described by traditional protein adsorption models. These coatings completely cover the surface with uniform, globular silk fibroin aggregates and are shown to be β-sheet rich (~40%) through ATR-FTIR analysis. However, photo-induced force microscopy and spectroscopy (PiF-IR), which provides sub-nm spatial resolution of protein conformations on the surface, shows evidence of a monolayer of β-sheet-rich protein chains distributed across the surface, while the globular aggregates are relatively β-sheet poor. This β-sheet-rich layer may serve as anchoring points to initiate growth of nano-thin silk fibroin coatings through protein-protein self-assembly. In contrast, chaotropic solution conditions that promote protein solubility, such as citric acid and sodium chloride solutions, do not allow for continuous, non-saturating growth of coatings. These results suggest that attractive protein-protein interactions are required for the growth of defect-free coatings tens of nanometers in thickness. Investigation of solution-phase self-assembly in phosphate concentrations that promote coating growth indicate that silk fibroin forms predominantly small aggregates approximately 30 nm in hydrodynamic diameter, which matches the size of globules found in the coating by AFM. At higher phosphate concentrations (>300 mM), where coating growth is inhibited, silk fibroin forms exclusively large, micron-sized aggregates in solution, which do not participate in coating growth. Thus, our findings suggest that the continuous growth of nanothin silk fibroin coatings by interfacial self-assembly requires an optimal balance between inter-protein assembly and surface adsorption.