JigSAP was dispersed in Dulbecco’s Modified Eagle Medium (DMEM containing 1.0 wt% HEPES, pH 7.4, 20 ☌). Molecular dynamics (MD) simulation indicated the one-dimensional self-assembly of JigSAP in an aqueous medium, suggesting nanofiber formation (Fig. The hydrophobic surface, with the sequence I–A–M–A–I, was designed based on the dovetail-packing domain in GYPA. 1a, ARMRA indicates the AX 1X 2X 3A motif X 1 and X 3: polar amino acids, X 2: non-polar amino acid), with alternating hydrophilic and hydrophobic amino acid residues, generating an amphiphilic structure by exposing hydrophilic and hydrophobic surfaces in the β-sheet. JigSAP has the sequence Ac-RIDARMRADIR-NH 2 (Fig. Inspired by the dynamic self-assembling properties of these natural sequences, we designed the jigsaw-shaped self-assembling peptide JigSAP containing the AXXXA motif. These motifs show α-helix-to-β-strand conformational transitions, generating a jigsaw-shaped hydrophobic surface that dimerizes with dovetail packing, which in turn results in nanofiber formation through β-sheet assembly.
Here, we show a cell-adhesive fiber-forming peptide that mimics the jigsaw-shaped hydrophobic surface in the dovetail-packing motif of glycophorin A (GYPA) 19, 20, called the jigsaw-shaped self-assembling peptide (JigSAP), allowed efficient incorporation and sustained release of vascular endothelial growth factor (VEGF), and showed cell transplantation-free regenerative therapeutic effects on a subacute-chronic phase mouse stroke model.ĪXXXA and GXXXG sequences are structural motifs found in homodimeric proteins such as glycophorin A (GYPA) 19, 20. However, incorporating and releasing secreted proteins are generally incompatible, and development of peptidic materials capable of incorporating and releasing secreted proteins remains mostly unexplored. Self-assembling peptide hydrogels can be widely used in clinical applications because of their cell-adhesive properties and degradability into chemically-defined molecules 18.
Various types of self-assembling peptidic materials have been reported as artificial ECMs 10, 11, 12, 13, 14, 15, 16, 17. These artificial ECMs are divided into chemically-crosslinked polymers and supramolecularly self-assembled molecules. Many artificial ECMs have been created for tissue engineering and regeneration 8, 9. However, after severe tissue injury, a secreted protein must be provided along with the ECMs. Attachment of these peptide domains to the C-termini of growth factors resulted in the engineered growth factors enhancing signal transduction from their receptors, and enhanced tissue repair 6, 7. For example, the peptide domains of placenta growth factor-2 (PIGF-2) and laminin α subunit have high affinity to ECMs. Covalently modified secreted proteins efficiently bind to and release from ECMs during injured tissue regeneration if the ECMs remain intact after injury. Covalent modification of cholesterol by hedgehog family proteins is essential for the morphogen gradient 3, 4 and is believed to regulate their binding to and release from heparan sulfate proteoglycans 5. For example, secreted proteins belonging to the hedgehog family interact with and dissociate from heparan sulfate proteoglycans and generate the morphogen gradient essential for proper development 3, 4. During development, ECMs regulate the binding and release of secreted proteins, and regulate cell adhesion 1, 2. This research highlights a therapeutic strategy for injured tissue regeneration using the jigsaw-shaped self-assembling peptide supramolecular hydrogel.Įxtracellular matrices (ECMs) are complex networks of proteins and other biomolecules, and provide a biological niche for regulating cellular responses such as proliferation, survival, and differentiation. The molecular- and macro-scale supramolecular properties of the jigsaw-shaped self-assembling peptide hydrogel allow efficient incorporation and sustained release of vascular endothelial growth factor, and demonstrate cell transplantation-free regenerative therapeutic effects in a subacute-chronic phase mouse stroke model. We show that the jigsaw-shaped self-assembling peptide forms several-micrometer-long supramolecular nanofibers through a helix-to-strand transition to afford a hydrogel under physiological conditions and disperses homogeneously in the hydrogel. Herein, we develop a cell-adhesive fiber-forming peptide that mimics the jigsaw-shaped hydrophobic surface in the dovetail-packing motif of glycophorin A as an artificial extracellular matrix for regenerative therapy. During injured tissue regeneration, the extracellular matrix plays a key role in controlling and coordinating various cellular events by binding and releasing secreted proteins in addition to promoting cell adhesion.