Probing Supramolecular Motion with Nuclear Magnetic Resonance in Bioactive Scaffolds that Promote Neural Regeneration in the Central Nervous System

Dynamics in supramolecular biomaterials have been recently identified as a critical feature in their in vivo performance based on cell signaling. This was originally observed in the context of peptide amphiphile (PA) assemblies that promote neural regeneration in the central nervous system, known to be an enormous challenge. Specifically, enhanced supramolecular motion controlled by the peptide sequence was found to reverse paralysis after trauma to the spinal cord, whereas slower dynamics failed to attain a similar outcome. Here, we report that the most bioactive large filamentous assemblies─bearing the laminin-derived IKVAV signal─exhibit the highest supramolecular dynamics, characterized for the first time by high-resolution magic-angle spinning NMR spectroscopy. For dynamic systems, we also report on 1 H and 13 C chemical shifts, 1 H– 1 H dipolar couplings, and 1 H relaxation data, which suggest pronounced β-sheet structuring within the IKVAV domain. Furthermore, dipolar coupling measurements suggest the possible formation of salt bridges between the E and K residues, folding of PA molecules, and the potential coexistence of parallel and antiparallel molecular arrangements within the dynamic assemblies. X-ray scattering and electron microscopy also revealed a significant population of small PA clusters in equilibrium with micron-length nanofilaments in the more dynamic, bioactive systems, consistent with our previous observations on a related supramolecular system. Based on DOSY NMR, these clusters have a hydrodynamic diameter of 6.6 nm, closely matching the 6.2 nm effective diameter from solution X-ray scattering. Finally, multiphase NMR unambiguously demonstrated the coassembly of PAs bearing the IKVAV moiety with those displaying a peptide that mimics fibroblast growth factor 2, which was necessary to reverse paralysis.