Abstract:

Objective The dissolution of silk fibroin protein disrupts its multi-order structure, leading to a decline in the mechanical properties of fibers, which in turn limits the application of micro-nano silk fibroin fiber membranes. By reconstructing the micro-mesoscopic structure of regenerated silk fibroin (RSF), this research aims to enhance the mechanical properties of RSF materials, thereby expanding their potential applications in the biomedical field.

Method In this study, we simulated the microenvironment within silkworm glands and induced liquid-liquid phase separation in the regenerated silk fibroin (RSF) solution through a salt ion system. Silk fibroin nanofibri-llars (SFNF) of various geometric dimensions were employed as reinforcements for the RSF material. By employing electrospinning, we fabricated mechanically enhanced, self-coagulating RSF micro-nano fiber membranes.

Results Sodium citrate (Na₃Citrate) solution was found the optimal system for inducing self-coagulation of RSF aqueous solutions. When the concentration of Na₃Citrate exceeded 0.6 mol/L and the concentration of the RSF solution was above 2%, the RSF aqueous solution began to undergo self-coagulation. This process intensified with increasing sodium citrate concentration. However, when the concentrations of both Na₃Citrate and RSF were excessively high (i.e., RSF above 16%, Na₃Citrate above 1.2 mol/L), the degree of self-coagulation became excessive, leading to the rapid formation of flaky precipitates within 20 minutes of solution preparation. With increasing concentration of Na₃Citrate, the entanglement of RSF macromolecular chains became more compact, leading to an increase in the β-sheet structure of the RSF solution from the initial 33.8% to 51.1%. This enhancement in internal flow resistance resulted in increased viscosity of the RSF solution, thereby improving its spinnability. At a Na₃Citrate concentration of 1.0 mol/L, a voltage of 22 kV, a flow rate of 0.2 mL/h, and a spinning distance of 16 cm, the fiber diameter and coefficient of variation (CV) were minimized, suggesting good spinning stability and a high specific surface area of the fibers. After incorporating SFNF of varying geometric dimensions, the spinning solution retained good spinnability. Compared to the RSF fiber membrane, the mechanical properties of the RSF-SFNF micro-nano fiber membrane were significantly enhanced. The tensile strength of RSF-SFNF₁₃₀ was increased from 0.46 MPa to 0.49 MPa, and the elongation at break of SF-SFNF₁₀₀ was improved from 2.06% to 3.54%. The ethanol treatment caused no significant changes on the surface of the fiber membrane. The content of β-sheet structure within the fiber membrane was increased to 50.0%, which ameliorated the solubility issue of RSF fiber membranes in water. The hemolysis rate was 2.58%, demonstrating good blood compatibility.

Conclusion Within the salt ion system, Na₃Citrate exhibits the most potent induction effect on the liquid-liquid phase separation of RSF. The β-sheet structure of the RSF solution increases from an initial 33.8% to 51.1%, which correspondingly enhances the overall viscosity and spinnability of the RSF solution. The incorporation of SFNF significantly improves the mechanical properties of RSF micro-nanofiber membranes, where the elongation at break is increased from 2.06% to 3.54%, and the tensile strength is elevated from 0.46 MPa to 0.49 MPa. Furthermore, the fiber membrane demonstrates good blood compatibility with a hemolysis rate of 2.58%, indicating promising potential for application in the field of wound dressing.

Key words: regenerated silk fibroin, self-coagulation, electrospinning, micro-nanofiber, mechanical reinforcement, biomedical material