A selection of self-assembling peptides (SAPs) based on the alternation of hydrophobic and hydrophilic amino acids: DABACABACD (A: hydrophobic residue: F phenylalanine or Y tyrosine, B/C: hydrophilic residue e.g.: K lysine or E glutamic acid) was designed. A parametric study was then carried out to verify the effect of rational peptide sequence modification on the final physicochemical properties of hydrogels. Tyramine-modified hyaluronan (THA) was also synthesized at two different molecular weights (280 kDa and 1640 kDa) and used to form composite hydrogels with self-assembling peptides. All parental peptides self-assembled into semi-flexible networks and hydrogels above critical gelation concentration in the region of 2.5-5 mM and display characteristic high β-sheet content. Self-assembly, rheological properties and printability of both peptide and peptide-THA hydrogels can be controlled by the choice of primary peptide sequence, fabrication technique and final crosslinking mechanisms including enzymatic (horseradish peroxidase, H2O2) and visible green light crosslinking using Eosin. These hydrogels are characterized by shear-thinning behavior and rapid recovery allowing extrusion-based fabrication of scaffolds. For the first time we also demonstrated the polarization effects of the supplemented THA on macrophages differentiated from human peripheral blood mononuclear cells over 5 days. M1 and M2 polarization modulated by the supplementation with low and high molecular weight THA were unraveled by the semi-automated image analysis from confocal microscopy, gene expression analysis and ELISA. Self-assembly, rheological properties and printability of SAPs were controlled by the choice of primary peptide sequence, concentration, and fabrication technique. Furthermore, the position of tyrosine in the peptide sequences dictated the distinct self-assembly into nanostructures, with sequences EF8 (control), EF8Y, YEF8Y, EYF8 self-assembling into thin fibers d<4 nm, whereas YEF8 self-assembling into rod-like flat ribbons d>20 nm. These distinct structural changes influenced the inflammatory profile, with peptides EF8 and EYF8 displaying anti-inflammatory profile, peptides EF8Y, YEF8Y remaining inert and peptide YEF8 displaying a pro-inflammatory state. In summary we uncover the link between basic molecular interactions driving self-assembly of tyrosine-containing SAPs into distinct nanostructures and demonstrate them as extrudable platforms for immunomodulatory tissue engineering. Finally, using traditional simplistic approach of “M1-like” as pro-inflammatory, and “M2-like” to as anti-inflammatory states, respectively, we confirm the typical association of low versus high molecular weight for chemically modified THA holds true and we thus bring immunomodulatory guidance towards the appropriate choice of it to a wider biomaterials and tissue engineering communities. We believe that these responses provide more rapid and accurate in vivo representation and will provide translational basis for the use of THA-based biomaterials in the future.
