Versatile Use of Tyramine-Modified Self-Assembling β-Sheet Peptides and Hyaluronic Acid Hydrogels—From Design and 3D Printing to Immunomodulation

Multifunctional biomaterials which exhibit well-defined physicochemical properties and encode spatiotemporally controlled biological signals have the potential to act as next generation advanced systems modulating cellular behaviour. Within tissue regeneration field one of the commonly arising themes is impaired regeneration associated with the compromised immune system.¹ One solution to tackle this issue is to design immunomodulatory biomaterials that temporally control the overall level of inflammation.¹ For example, it has been shown that molecular weight of hyaluronic acid (HA) directly influences the polarization state of macrophages.² Designing functional materials that incorporate immunomodulatory effects, biocompatibility and that allow stable long-term polarization of macrophages is therefore of high interest in tissue engineering and 3D bioprinting.¹<br/>Self-assembling β-sheet forming peptides and tyramine-modified HA (THA) are two examples of biopolymers that have been shown as vital biomaterials spanning advanced cell culture systems up to effective minimally complex microenvironments for the generation of organoids.^{3, 4} The role of these materials interfacing immune cells is not yet fully uncovered, therefore here we designed a selection of hydrogels built from self-assembling β-sheet forming peptides⁵ and THA,⁶ that can be processed by 3D micro-extrusion printing. A selection of peptide sequences was based on the alternation of hydrophobic and hydrophilic amino acids: <b>D</b><b>A</b><b>B</b><b>A</b><b>C</b><b>A</b><b>B</b><b>A</b><b>C</b><b>D</b> (<b>A</b>: hydrophobic residue: <b>F</b> phenylalanine or <b>Y</b> tyrosine, <b>B</b>/<b>C</b>: hydrophilic residue e.g.: <b>K</b> lysine or <b>E</b> glutamic acid). THA of two molecular weights (280 kDa and 1640 kDa) were synthesized as previously described.⁶<br/>A parametric study was carried out on the designed selection of <b>D</b><b>A</b><b>B</b><b>A</b><b>C</b><b>A</b><b>B</b><b>A</b><b>C</b><b>D</b> peptides to verify the effect of rational peptide sequence modification on final physicochemical properties of peptide alone and peptide-THA composite hydrogels. <b>D </b>residues were rationally varied between hydrophobic (<b>Y</b>) or hydrophilic (<b>E</b>) amino acids to modulate the interactions' ability of formed β-sheet edges and shell with other peptide fibres and THA. 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. For the first time we also demonstrate 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 unravelled by the semi-automated image analysis from confocal microscopy, gene expression analysis and ELISA.<br/>In summary here we uncover the link between basic molecular interactions driving self-assembly of functional tyramine modified peptide-based and hyualuronic acid-based biomaterials and demonstrate their capabilities as extrudable platforms for immunomodulatory tissue engineering.<br/><b>Acknowledgements</b><br/>This work was supported by the European Union’s Horizon 2020 (H2020-MSCA-IF-2019) research and innovation programme under the Marie Sklodowska-Curie grant agreement 893099 — ImmunoBioInks.<br/><b>References</b><br/>1. C. M. Walsh, <i>et al.</i>, Pharmacology & Therapeutics, 2022, 234, 108043.<br/>2. J. E. Rayahin, <i>et al.</i>, ACS Biomaterials Science & Engineering, 2015, 1, 481-493.<br/>3. A. Schwab, <i>et al., </i>Materials Today Bio, 2020, 7, 100058.<br/>4. N. J. Treacy, <i>et al.</i>, Bioactive Materials, 2023, 21, 142-156.<br/>5. J. K. Wychowaniec, <i>et al., </i>Biomacromolecules, 2020, 21, 2285-2297.<br/>6. C. Loebel, <i>et al.</i>, Biomacromolecules, 2017, 18, 855-864.