Specific Ion Effects to Modulate Free-Standing Thin Silk Fibroin Films

Introduction: Thin polymeric films, with thicknesses below one micrometer, have massive potential in various biomedical applications, partly due to their thinness-related properties. One example is the thin film used in lung-on-a-chip to recapitulate the essential tissue interface between the endothelium and epithelium of the lung alveoli. However, such films usually face notable challenges in recapitulating the native physiological composition in the natural cellular environment, thus restricting potential biomedical applications and underscoring a need for alternative materials. Here, we demonstrate salt ions-mediated and spin-coated thin films in silk fibroin (SF), a structural protein derived from Bombyx mori silkworms, as a viable alternative to the current thin polymeric films. The proteinaceous composition and the salt ion-based regulation of SF films are advantageous in the recapitulation of the essential tissue interface.

Methods: Free-standing SF films were fabricated using a spin coating approach and a sacrificial layer of 10% (w/v) dextran beneath the SF film. The spinning speed, ranging from 1,500 to 12,000 rpm, was used to control the thickness of the SF films. After spin coating, the film is treated with one of several different salt solutions, including ammonium sulfate, potassium phosphate, and sodium chloride. The water-soluble dextran layer was removed by immersion in an aqueous solution, resulting in free-standing SF film floating on the solution surface. A homemade tool was developed to extract the films while maintaining their integrity. The morphology, structure, and the relation between the thickness of SF films and processing conditions, such as spinning speed and salt composition, were investigated using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and spectroscopic ellipsometry (SE). SE measurement is based on polarization changes (Psi (Ψ) and Delta (Δ)) of the reflected light as it interacted with films of varying thicknesses. The data were fitted using the Cauchy dispersion model to obtain thickness-relevant coefficients.

Results and Discussion: Analysis of SE measurements indicates that the thin thickness of spin-coated SF films, ranging from approximately 700 nm to 100 nm, can be tuned using spinning speeds and salt ion treatments. Notably, the thickness of SF films is inversely related to the spinning speed. The trend of thickness is consistent in both SEM and SE. While the measurements from SEM are always lower than SE, it may be attributed to different approaches to preparing samples. The obtained hundreds of nanometers thickness enables a closer resemblance to the basement membrane in the natural tissue interfaces. FTIR spectra indicate that salt ion treatments significantly influenced the secondary structure of SF films. Specifically, the ammonium sulfate and potassium phosphate treatments resulted in high β-sheets content (approximately 44–46%) and a continuous and dense film morphology observed in SEM images. In contrast, sodium chloride treatment increased α-helix-like content to about 40% and induced a porous and network morphology. The tunability of film morphology through salt ion treatment could prove advantageous for tissue engineering applications, where specific structural properties are essential for supporting cell adhesion and proliferation.

Conclusions: Our study establishes an innovative fabrication method for free-standing thin SF films characterized by salt ion-mediated morphology. This result implies huge potential in reconstructing the tissue interface between endothelium and epithelium for lung on a chip.