Introduction: Neurodegenerative diseases affect millions worldwide and remain without effective regenerative treatments. Advances in neuroprosthetics and bioelectronic medicine increasingly rely on materials capable of interfacing with neural tissue and supporting functional recovery. Semiconductive polymers offer bioelectrical and structural cues that can modulate neuronal behavior without exogenous stimulation, making them promising candidates for regenerative strategies. This study investigated the ability of poly(3‑hexylthiophene) (P3HT)-coated substrates to promote spontaneous neuronal differentiation, maturation, and synaptic network formation in human SH‑SY5Y neuroblastoma cells cultured under non‑inductive conditions.
Methods: The surface potential of P3HT films was characterized using PeakForce Kelvin Probe Force Microscopy (PF‑KPFM), with ΔCPD values calculated relative to highly oriented pyrolytic graphite to ensure tip‑independent measurements. SH‑SY5Y cells were seeded on P3HT‑coated or uncoated control slides and maintained for 15 days. Cell viability and proliferation were assessed using MTS assays and DAPI‑based nuclear quantification. Neuronal differentiation was evaluated through qPCR analysis of cytoskeletal and synaptic genes (SOX2, NES, TUBB3, SYN, SYP), complemented by immunofluorescence staining for TUBB3, MAP2, and NFH. Neurite outgrowth was quantified using NeuronJ, and functional relationships among neuronal markers were explored using STRING‑based protein–protein interaction analysis.
Results: P3HT substrates demonstrated excellent biocompatibility and significantly enhanced SH‑SY5Y viability and proliferation over time. Gene expression and immunofluorescence analyses revealed increased levels of neuronal cytoskeletal and synaptic markers, accompanied by pronounced neurite elongation and branching. These findings indicate that P3HT promotes spontaneous neuronal differentiation and supports the emergence of early synaptic networks even in the absence of chemical inducers.
Conclusions: P3HT‑based interfaces act as potent modulators of neuronal maturation and network formation, highlighting their potential for next‑generation neural interfaces and regenerative bioelectronic applications.
