The delivery of protein therapeutics in a non-invasive, safe, and sustained manner, without reliance on viral delivery systems, is critical for treating a wide range of chronic injuries and diseases. Among the many challenges, achieving axonal regeneration and functional restoration after CNS injury or disease remains elusive for most clinical interventions, which continues to drive the need for innovative solutions. In this study, a thermoresponsive hydrogel system based on recombinant spider silk protein (fibroin) was developed. The protein solution undergoes a rapid sol-gel transition upon elevated temperature (37°C) after brief sonication. This thermally triggered gelation renders the system injectable. Using SpyTag/SpyCatcher chemistry, hydrogels composed of SpyTag-fused fibroin can be functionalized with various SpyCatcher-fused bioactive motifs, such as neurotrophic factors (e.g., ciliary neurotrophic factor) and cell-binding ligands (e.g., laminin), making them ideal for neuronal culture. More importantly, intravitreal injection of protein materials decorated with SpyCatcher-fused CNTF into the vitreous after optic nerve injury can lead to sustained activation of the JAK/STAT3 signaling pathway, improved neuronal survival, and enhanced axonal regeneration. This study demonstrates a universal material system for injectable, sustained delivery of protein therapeutics for neuroprotection and regeneration, and may be extended to the treatment of other chronic diseases and injuries.

Innovations:
1. A new thermoresponsive hydrogel system based on recombinant spider silk was developed, which achieved rapid gelation at physiological temperature.
2. The SpyTag/SpyCatcher chemical reaction was cleverly used to achieve multifunctional modification of hydrogels, allowing the system to carry neurotrophic factors and cell adhesion ligands at the same time.
3. The long-term sustained release of neurotrophic factors was successfully achieved, promoting neuronal survival and axonal regeneration by continuously activating the JAK/STAT3 signaling pathway.

Inspiration from scientific research:
1. Material design should fully consider the actual needs of clinical applications and integrate injectability, biocompatibility and functionality. This systematic thinking has important guiding significance for the development of new biomaterials.
2. In the process of material development, we are good at using biochemical tools (such as SpyTag/SpyCatcher system) to realize the functional modification of materials. This interdisciplinary research idea can often bring breakthrough progress.

3. The therapeutic effect of materials is verified by regulating signal pathways. This molecular-level mechanism research can not only prove the effectiveness of materials, but also provide a theoretical basis for subsequent optimization.

Extension of ideas:
1. This system is expected to be further expanded to other types of central nervous system diseases, such as spinal cord injury, Alzheimer's disease, etc., to achieve disease treatment by delivering different therapeutic proteins.
2. The SpyTag/SpyCatcher chemical modification strategy can be extended to other types of biomaterial systems to build multifunctional drug delivery platforms and achieve more complex therapeutic goals.
3. The design concept of this thermoresponsive hydrogel can inspire the development of other intelligent responsive materials, such as pH response, enzyme response, etc., to meet the needs of different disease treatments.

4. This study provides a paradigm for the development of new protein drug delivery systems. This non-viral vector system has obvious advantages in safety and controllability and is worth exploring in other therapeutic areas.