SUPRAMOLECULAR NANOCOMPOSITE HYDROGELS FOR NERVE TISSUE REGENERATION

The regeneration of nerve tissue remains one of the most formidable challenges in modern biomedicine due to the limited self-repair ability of neurons and the mechanical and electrical complexity of neural environments. This work explores an integrated strategy combining functionalized carbon nanotubes (f-CNTs) and self-assembling peptide hydrogels to create multifunctional nanocomposite scaffolds that mimic the extracellular matrix, while enabling electrical stimulation and biochemical support for neuronal growth. Multi-walled CNTs (MWCNTs) were selected for their high conductivity and mechanical strength, although they are hydrophobic and poorly dispersible. To overcome these limitations, a tyrosine (Tyr)-based derivative was synthesized and covalently grafted onto CNTs using the Tour reaction. Presence of Tyr on f-CNTs could enable favorable interactions with Try-containing self-assembling peptides within nanocomposite gels, and also open the way for covalent crosslinking. To this end, three heterochiral tripeptides with the diphenylalanine self-assembling motif, and Tyr in different positions along the sequence (i.e., yFF, fYF, and fFY) were designed, prepared, and characterized. The influence of Tyr position was assessed on self-assembly, gelation, and photo-crosslinking reactivity. Rheological measures revealed that all three formed supramolecular hydrogels, with fFY producing the most mechanically robust network. Riboflavin-mediated photo-crosslinking of Tyr residues was then investigated as a sustainable strategy to reinforce the soft hydrogel network. Spectroscopic and chromatographic monitoring identified optimal irradiation conditions leading to di-Tyr formation. The reaction was then tested to reinforce the three hydrogels. In particular, a successful crosslinking was attained for fFY, while gel rupture was observed for yFF. Our results obtained via spectroscopic and computational techniques enabled to postulate a hypothesis for the rational explanation of the observed outcomes of the photo-crosslinking reaction, based on different peptide conformation and Tyr exposure to solvent. Furthermore, two peptides containing the cell-adhesive sequences LDV and IKVAV were synthesized and incorporated within the hydrogel to promote integrin-mediated cell adhesion. However, despite both peptides displayed very good cytocompatibility and activity, the IKVAV-containing peptide displayed solubility issues that prevented further testing. The final nanocomposite scaffold was then assembled and characterized via rheological and microscopic analyses, and was finally loaded with a small-molecule Brain-Derived Neurotrophic Factor (BDNF) mimetic to stimulate neuronal differentiation. Controlled-release assays confirmed sustained diffusion of the mimetic over 9 days. Preliminary in vitro studies on neuroblastoma cells demonstrated the ability of the nanocomposite material to induce the differentiation of these cells, but also highlighted a marked (photo)toxicity of its components, unveiling both the need for further optimization of experimental conditions, but also the possibility of an anticancer activity. This thesis therefore demonstrated the effectiveness of Tyr photo-crosslinking on self-assembling tripeptides, emphasizing that both molecular sequence and supramolecular arrangement critically influence the crosslinking outcome. The presence of Tyr was also used to link f-CNTs with the self-assembling tripeptides in the nanocomposite hydrogels. The developed nanocomposite hydrogels exhibited improved mechanical stability and promising performance in preliminary tests on neuroblastoma cells, supporting their potential for neural tissue regeneration. Interestingly, the photo-crosslinked material also displayed intrinsic anticancer activity, suggesting future exploration as a multifunctional scaffold capable of promoting healthy cells regeneration, while modulating oxidative stress in tumour environments.