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

Abstract

Peptides are of continued interest for therapeutic applications, from soluble and immobilized ligands that promote desired binding or uptake to self-assembled supramolecular structures that serve as scaffolds in vitro and in vivo. These applications require efficient and scalable synthetic approaches because of the large amounts of material that often are needed for studies of bulk material properties and their translation. In this work, we establish new methods for the synthesis, purification, and visualization of assembling peptides, with a focus on multifunctional collagen mimetic peptides (mfCMPs) relevant for formation and integration within hydrogel-based biomaterials. First, a methodical approach useful for the microwave-assisted synthesis of assembling peptide sequences prone to deletions was established, beginning with the identification of the deleted residues and their locations and followed by targeted use of dual chemistry couplings for those specific residues. Second, purification techniques that integrate the principles of heating and ion displacement with traditional chromatography and dialysis were implemented to improve separation and isolation of the desired multifunctional peptide product, which contained blocks for thermoresponsiveness and ionic interactions. Third, an approach for fluorescent labeling of these mfCMPs, which is orthogonal to their assembly and their covalent incorporation into a bulk hydrogel material, was established, allowing visualization of the resulting hierarchical fibrillar structures in three dimensions within hydrogels using confocal microscopy. The methods presented in this work allow the production of multifunctional peptides in scalable quantities and with minimal deletions, enabling future studies for better understanding of composition−structure−property relationships and for translating these biomaterials into a range of applications. Although mfCMPs are the focus of this work, the methods demonstrated could prove useful for other assembling peptide systems and for the production of peptides more broadly for therapeutic applications.

Low-power microwaves: a cell-compatible physical treatment to enhance the mechanical properties of self-assembling peptides

“…67 Recently, a discussion was presented by Ford and Kloxin regarding the use of MW heating in the synthesis of SAPs, in the disruption of peptide aggregation, in the formation of but β-aggregates, and in overcoming the steric hindrance. 68…”

“…ThT is a dye used to detect β-rich structures, 68 since it increases fluorescence upon interacting with β-sheet-rich peptides and proteins, its fluorescence intensity is, therefore, indicative of samples with β-sheet content. 69 ThT assay showed β-structuration in all samples (amyloid-binding emission peak at 490 nm), but the fluorescence intensity increased after MW treatments by varying the time and power energy, reaching the highest peaks for 20 min treatment at 1 W in both BM3 and HYDROSAP (Fig.…”

Editorial: Special Issue on Advanced Biomedical Hydrogels

“…This special issue encompasses new methods toward these goals, which will influence the success of hydrogels. Beginning with the synthesis of new molecules to assemble into biomedical hydrogels, Ford and Kloxin report methods for the synthesis, purification, and visualization of assembling peptides with a focus on collagen mimetic peptides . These methods support the scalable production of peptides that allow for the investigation of structure–property relationships and the translation of self-assembled biomaterials.…”

Linguistic Analysis Identifies Emergent Biomaterial Fabrication Trends for Orthopaedic Applications

“…Across all orthopedic tissue types, multi-scale mechanical testing of biomaterials/constructs has become the standard, with assays from the macroscale to the nanoscale required to assess anatomical properties and gradients (Figure 7a). The choice of structural elements, including collagen (and collagen mimics [54] ), glycosaminoglycan (and proteoglycan mimics [73] ), and mineralization content [62,74] influence the final construct structural properties, as do alignment, topography, and porosity (Figure 7a). In many tissues, the combination of local mechanical properties and their structural organization is key to matching native tissue strength and architecture (Figure 7a).…”

“…[ 18 ] Material designs range from single‐function materials that support endogenous repair to more complex combinations of multi‐functional materials for the delivery of exogenous cells or the provision of biochemical signals that promote various aspects of tissue repair. [ 54 ] The inclusion of bioactive cues, either spatial, biochemical, or mechanical, may provide important guides for directed healing; however, the extent of biomimicry necessary to elicit tissue regeneration remains unclear. From the perspectives of fabrication and commercialization, simplicity of design and use of materials that are already Food and Drug Administration (FDA)‐approved may help to reduce cost and regulatory requirements for translation.…”