We have developed a class of self-assembling peptides that undergo triggered hydrogelation in response to physiological conditions to afford mechanically rigid, viscoelastic hydrogels. These gels display shear-thin recovery behavior that allows them to be syringe delivered. Peptides that comprise a given gel are small (20 residues), de novo designed beta-hairpins composed of two beta-strands of alternating hydrophobic and hydrophobic residues. A tetrapeptide (-VdPPT-) sequence with a high propensity to form a type II beta-turn centrally connects the two strands. In low ionic strength, aqueous solutions, peptides are freely soluble and remain unfolded due to electrostatic repulsions between the hydrophilic residues. Increasing the ionic strength of the solution by adjusting the NaCl concentration to 150 mM (physiologically relevant salt concentration) screens some of this charge and promotes the folding of the peptides into facially amphiphilic beta-hairpin structures. In addition to adjusting the ionic strength, increasing the temperature also triggers folding by driving the hydrophobic effect. Folded hairpins subsequently self assemble to form a physically crosslinked network of fibrils. Although discussed as separate events, biophysical analysis suggests that the folding and self-assembly events are linked equilibria. Small angle neutron scattering (SANS) and transmission electron microscopy (TEM) data show that the hairpins self-assemble laterally by forming a network of intermolecular hydrogen bonds that define the long axis of a given fibril;all the beta-strands of the assembled hairpins are in register affording fibrils of distinct diameter ( 3 nm). Peptides also assemble in a facial manner by burying their hydrophobic residues to form a bilayer that defines the thickness of a given fibril as demonstrated by AFM. Along the long axis of a given fibril, bilayer formation occurs in a regular fashion with one hairpin docked, and in register, with its partner. This arrangement shields the maximal amount of hydrophobic surface area from water. However, as shown by cryo-TEM and oscillatory rheology, imperfections in this mechanism occur where the face of one hairpin is rotated relative to its partner in the bilayer. This results in a site for nascent fibril growth in a new three-dimensional direction and constitutes the formation of an inter-fibril crosslink. These inter-fibril crosslinks, in addition to fibril entanglements, are physical crosslinks that define the mechanical rigidity of a given gel. By linking the triggered folding event to self-assembly, hydrogel materials can be made with temporal and spatial resolution. We have shown that peptide folding and assembly can be triggered in the presence of polysaccharides and proteins to directly encapsulate these macromolecules within the resulting hydrogel network. The resulting loaded gel can be easily delivered to a secondary site via syringe where it can subsequently release its payload. In addition, hydrogelation can be triggered in the presence of mammalian cells affording gels loaded with cells that can be delivered by syringe to in vivo sites;this technology holds promise for site-specific tissue engineering and spatially resolved cytomedical therapy. Recently we have: 1. Characterized the mass transport properties of several gel compositions with respect to polysaccharide and protein diffusion and delivery. 2. Developed photochemical methods to enhance the setting properties of the gels. 3. Developed peptides that undergo domain swapping during hydrogelation to provide control over fibril nanostructure. 4. Expanded our mechanistic understanding of the folding and self-assembly events. 5. Established sequence-material properties relationships for the hydrophobic face of the hairpin. We are currently: 1. Establishing cytocompatibility profiles using mammalian cells that have been encapsulated and shear-thin delivered. 2. Investigating distinct cell-material interactions that influence cell fate employing model chondrocytes and NON-embryonic stem cells. 3. Developing general synthetic protein-based crosslinking strategies to prepare highly functional materials. 4. Developing synthetic strategies to prepare glycosylated, self-assembled materials. 5. Developing smart vesicles for thermally-triggered drug delivery. 6. Developing stereochemical methods to control bulk material properties. 7. Developing methods to control hydrogel biodegradation. 8. Developing bacterial expression systems to facilitate material production. This basic science ultimately leads to the discovery of novel materials for biomedical use.
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