Mechanistic Insights into Hyaluronic Acid Induced Peptide Nanofiber Organization in Supramolecular Hydrogels

Composite hydrogels composed of low-molecular-weight peptide self-assemblies and polysaccharides are gaining great interest as new types of biomaterials. Interactions between polysaccharides and peptide self-assemblies are well reported, but a molecular picture of their impact on the resulting material is still missing. Using the phosphorylated tripeptide precursor Fmoc-FFpY (Fmoc, fluorenylmethyloxycarbonyl; F, phenylalanine; Y, tyrosine; p, phosphate group), we investigated how hyaluronic acid (HA) influences the enzyme-assisted self-assembly of Fmoc-FFY generated in situ in the presence of alkaline phosphatase (AP). In the absence of HA, Fmoc-FFY peptides are known to self-assemble in nanometer thick and micrometer long fibers. The presence of HA leads to the spontaneous formation of bundles of several micrometers thickness. Using fluorescence recovery after photobleaching (FRAP), we find that in the bundles both (i) HA colocalizes with the peptide self-assemblies and (ii) its presence in the bundles is highly dynamic. The attractive interaction between negatively charged peptide fibers and negatively charged HA chains is explained through molecular dynamic simulations that show the existence of hydrogen bonds. Whereas the Fmoc-FFY peptide self-assembly itself is not affected by the presence of HA, this polysaccharide organizes the peptide nanofibers in a nematic phase visible by small-angle X-ray scattering (SAXS). The mean distance d between the nanofibers decreases by increasing the HA concentration c, but remains always larger than the diameter of the peptide nanofibers, indicating that they do not interact directly with each other. At a high enough HA concentration, the nematic organization transforms into an ordered 2D hexagonal columnar phase with a nanofiber distance d of 117 angstrom. Depletion interaction generated by the polysaccharides can explain the experimental power law variation d similar to c (-1/4) and is responsible for the bundle formation and organization. Such behavior is thus suggested for the first time on nano-objects using polymers partially adsorbing on self-assembled peptide nanofibers.

Automated summary

Composite hydrogels composed of low-molecular-weight peptide self-assemblies and polysaccharides have attracted great interest as new biomaterials. In this study, the impact of hyaluronic acid (HA) on the enzyme-assisted self-assembly of Fmoc-FFY peptides was investigated. HA was found to induce the formation of bundled peptide nanofibers, and the presence of HA in the bundles was dynamic. Molecular dynamic simulations explained the attractive interaction between HA and the peptide fibers through hydrogen bonds. The presence of HA also led to the organization of the peptide nanofibers into a nematic phase and an ordered 2D hexagonal columnar phase.