Enzyme-responsive release of Insulin-like Growth Factor-1 from recombinant spider-silk protein nanofibrous membrane for enhanced neural cell proliferation

Abstract

The application of biomaterial scaffolds capable of controllable growth factor release plays a pivotal role in nerve tissue repair. In this study, a recombinant fusion protein RSSP@IGF was designed and fabricated, yielding a novel recombinant spider silk protein nanofiber membrane. By integrating insulin-like growth factor-1 (IGF-1) with recombinant spider silk protein (RSSP) and incorporating specific cleavage sites responsive to thrombin and TEV protease, the construct enabled the controlled and sustained delivery of IGF-1. It was also expressed and purified using the Escherichia coli system to obtain a purity exceeding 90% RSSP@IGF. Subsequently, uniform nanofiber membranes were prepared by electrospinning. The membrane delivered excellent biocompatibility, with a hemolytic activity of 0.3%, and could significantly promote the growth of PC12 cells. Its degradation kinetics could be precisely adjusted by thrombin concentration, and 20–90% degradation achieved within 30 min at concentrations ranging from 0.1 to 4.0 U/mL. The IGF-1 release profile triggered by TEV protease showed that following its initial rapid release phase (0–24 h), its biological activity level could be maintained for up to 120 h. Notably, compared with all control groups (tissue culture plate, RSSP alone, and RSSP with soluble IGF-1), the membrane significantly enhanced PC12 cell proliferation within 10 days, demonstrating the advantage of the local delivery system. Mechanism studies revealed that RSSP@IGF may promote the proliferation of PC12 cells via the PI3K/Akt/mTOR signaling pathway. In summary, the controllable degradation spider silk protein nanofiber membrane constructed in this article provides a highly biocompatible and efficient platform for the sustained delivery of IGF-1. Its controllable degradation and sustained release characteristics hold enormous potential for applications in nerve regeneration and other fields warranting precise spatiotemporal control of growth factor presentation.

Graphical Abstract