Photo-triggerable laminin mimetic peptides for directional neural regeneration
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Abstract
The restoration of neuronal activity after injury or during aging requires neuron repopulation at the site of injury, directional regeneration of new nerves and oriented generation of new synapses. The ECM protein Laminin is abundant in neuronal microenvironment and is known to be involved in directing neuronal migration, differentiation and neurite development. In this thesis, a strategy for in vitro directional neurite growth in soft hydrogels is presented. It is based on the spatiotemporal control of the availability of Laminin adhesive motifs within synthetic hydrogels using light as an external guiding trigger.
Different variants of Laminin mimetic peptides containing the IKVAV were selected as ligands to mediate control over axonal growth on biomaterials. The photo-cleavable groups 3-(4,5-dimethoxy-2-nitrophenyl)-2-butanol (DMNPB), 6-nitroveratryl alcohol (NVOC) and 2,2'-((3'-(1-hydroxypropan-2-yl)-4'-nitro-[1,1'-biphenyl]-4-yl)azanediyl)bis(ethan-1-ol) (HANBP) were inserted at the K rest of the peptide to temporally block IKVAV bioactivity. Poly(acrylamide) (PAAm) hydrogel films with varied stiffness from 0.2-70 kPa were used as 2D substrates to study IKVAV-guided directional growth of axons. Two novel acryl monomers carrying methylsulfone (MS) side chains were developed to tune specific coupling of thiol terminated IKVAV to the PAAm gel at physiological conditions. The ability of the photoactivatable IKVAV-containing peptides to trigger and support neurite outgrowth was studied and compared in different cell biology experiments using neural progenitor cells from mouse embryo. The ability of the photoactivatable IKVAV-containing peptides to trigger and support spatial organization of neurons was demonstrated by using masked irradiation. The in-situ light exposure of IK(HANBP)VAV by scanning lasers allowed spatially directed neurite development in 2D cell cultures.
In the last part of the Thesis, an attempt to extend the photoactivation strategy to 3D environments was made by using two-photon activatable chromophores. The p-methoxynitrobiphenyl (PMNB) photoremovable group was introduced at aspartic acid residue of RGD sequence, a common adhesive motif used for cell attachment to biomaterials. Degradable hydrogels modified with RGD(PMNB)fC peptide were developed and 3D resolved spatial photoactivation inside the gel using two-photon laser guided migration of fibroblasts L929 within the 3D network was established. These results demonstrate that photoactivatable adhesive peptides can be used for spatiotemporal activation of attachment, migration and directional growth of cells in 2D and 3D cultures and provide a tool to control and pattern cell processes in relevant biomedical applications.