The optimized PBAE-loaded pro-neurogenic IFs nanoparticles reprogram astrocytes within the glial scar into diverse neuronal subtypes

Glial scar formation and neuronal loss following spinal cord injury (SCI) are major impediments to neural regeneration. Therefore, remodeling the inhibitory scar obstacles and replenishing lost neurons are paramount for neural repair and functional recovery. However, current prevailing therapeutic approaches faced formidable challenges in concurrently achieving effective neuronal replenishment and glial scar (GS) remodeling. Herein, we described an innovative “remodeling-regeneration integrated” non-viral therapeutic strategy designed to directly reprogram reactive astrocytes within GS into functional neurons of distinct subtypes in vitro. This reprogramming is achieved by utilizing poly(β-amino ester) (PBAE) nanoparticles for the efficient co-delivery of the proneural transcription factors ASCL1 and NGN2 (TFs-A/N) into glial scar cells. For the delivery of two genes, the PBAE polymer was synthesized under an optimized Michael addition reaction condition. Our results demonstrated that this polymer possesses low toxicity, favorable biodegradability, and high molecular weight, allowing efficient encapsulation of substantial amounts of TFs-A/N-encoding plasmids into stable nanoparticles. Importantly, these optimized nanoparticles exhibited high gene delivery efficiency. Of particular significance, PBAE-TFs-A/N nanoparticles not only effectively remodeled GS architecture but also induced the generation of diverse neuronal subtypes, including cholinergic, glutamatergic, GABAergic, and dopaminergic neurons, as evidenced by subtype-specific biochemical marker expression. Furthermore, the converted cells acquired neuronal functionality, demonstrated by expression of synaptic markers (SYN and PSD95), action potential firing, and neuron-specific sodium and potassium currents. These findings provide compelling evidence for a safe and effective non-viral reprogramming methodology that simultaneously promotes multi-subtype neuron generation and GS remodeling. Collectively, the study positions the approach as a promising avenue for novel insights into CNS injury repair and clinical translational applications.