Rapid Production of Multifunctional Self-Assembling\nPeptides for Incorporation and Visualization within Hydrogel Biomaterials

Abstract

Peptides\nare of continued interest for therapeutic applications,\nfrom soluble and immobilized ligands that promote desired binding\nor uptake to self-assembled supramolecular structures that serve as\nscaffolds <i>in vitro</i> and <i>in vivo</i>.\nThese applications require efficient and scalable synthetic approaches\nbecause of the large amounts of material that often are needed for\nstudies of bulk material properties and their translation. In this\nwork, we establish new methods for the synthesis, purification, and\nvisualization of assembling peptides, with a focus on multifunctional\ncollagen mimetic peptides (mfCMPs) relevant for formation and integration\nwithin hydrogel-based biomaterials. First, a methodical approach useful\nfor the microwave-assisted synthesis of assembling peptide sequences\nprone to deletions was established, beginning with the identification\nof the deleted residues and their locations and followed by targeted\nuse of dual chemistry couplings for those specific residues. Second,\npurification techniques that integrate the principles of heating and\nion displacement with traditional chromatography and dialysis were\nimplemented to improve separation and isolation of the desired multifunctional\npeptide product, which contained blocks for thermoresponsiveness and\nionic interactions. Third, an approach for fluorescent labeling of\nthese mfCMPs, which is orthogonal to their assembly and their covalent\nincorporation into a bulk hydrogel material, was established, allowing\nvisualization of the resulting hierarchical fibrillar structures in\nthree dimensions within hydrogels using confocal microscopy. The methods\npresented in this work allow the production of multifunctional peptides\nin scalable quantities and with minimal deletions, enabling future\nstudies for better understanding of composition–structure–property\nrelationships and for translating these biomaterials into a range\nof applications. Although mfCMPs are the focus of this work, the methods\ndemonstrated could prove useful for other assembling peptide systems\nand for the production of peptides more broadly for therapeutic applications.