Folding, self-assembly, and bulk material properties of a de novo designed three-stranded beta-sheet hydrogel.

A de novo designed three-stranded beta-sheet (TSS1) has been prepared that undergoes temperature-induced folding and self-assembly to afford a network of beta-sheet rich fibrils that constitutes a mechanically rigid hydrogel. Circular dichroism and infrared spectroscopies show that TSS1 folds and self-assembles into a beta-sheet secondary structure in response to temperature. Rheological measurements show that the resulting hydrogels are mechanically rigid [at pH 9, G' = 1750-9000 Pa, and at pH 7.4, G' = 8500 Pa] and that the storage modulus can be modulated by temperature and peptide concentration. Nanoscale structure analysis by transmission electron microscopy and small angle neutron scattering indicate that the hydrogel network is comprised of fibrils that are about 3 nm in width, consistent with the width of TSS1 in the folded state. A unique property of the TSS1 hydrogel is its ability to shear-thin into a low viscosity gel upon application of shear stress and immediately recover its mechanical rigidity upon termination of stress. This attribute allows the hydrogel to be delivered via syringe to a target site with spatial and temporal resolution. Finally, experiments employing C3H10t1/2 mesenchymal stem cells seeded onto the hydrogel and incubated for 24 h indicate that the TSS1 hydrogel surface is noncytotoxic, supports cell adhesion, and allows cell migration.

In vitro assessment of the pro-inflammatory potential of beta-hairpin peptide hydrogels.

The pro-inflammatory potential of beta-hairpin peptide hydrogels (MAX1 and MAX8) was assessed in vitro by measuring the cellular response of J774 mouse peritoneal macrophages cultured on the hydrogel surfaces. An enzyme-linked immunosorbent assay (ELISA) was used to measure the level of TNF-alpha, a pro-inflammatory cytokine, secreted by cells cultured on the gel surfaces. Both bulk and thin films of gels did not elicit TNF-alpha secretion from the macrophages. In addition, live/dead assays employing laser scanning confocal microscopy (LSCM) and phase-contrast light micrographs show the hydrogel surfaces are non-cytotoxic toward the macrophages and allow the cells to adopt healthy morphologies. When macrophages were activated with lipopolysaccharide (LPS), a known bacterial pathogen that activates an innate immune response, an increase in the TNF-alpha titers by two orders of magnitude was observed. On LPS induction, macrophages displayed a decrease in cell density, enlarged nuclei, and an increase in cytoplasmic granularity, all characteristics of activated macrophages indicating that the cells are still capable of reacting to insult. The data presented herein indicate that MAX1 and MAX8 gels do not elicit macrophage activation in vitro and suggest that these materials are excellent candidates for in vivo assessment in appropriate animal models.

Inherent antibacterial activity of a peptide-based beta-hairpin hydrogel.

Among several important considerations for implantation of a biomaterial, a main concern is the introduction of infection. We have designed a hydrogel scaffold from the self-assembling peptide, MAX1, for tissue regeneration applications whose surface exhibits inherent antibacterial activity. In experiments where MAX1 gels are challenged with bacterial solutions ranging in concentrations from 2 x 10(3) colony forming units (CFUs)/dm2 to 2 x 10(9) CFUs/dm2, gel surfaces exhibit broad-spectrum antibacterial activity. Results show that the hydrogel surface is active against Gram-positive (Staphylococcus epidermidis, Staphylococcus aureus, and Streptococcus pyogenes) and Gram-negative (Klebsiella pneumoniae and Escherichia coli) bacteria, all prevalent in hospital settings. Live-dead assays employing laser scanning confocal microscopy show that bacteria are killed when they engage the surface. In addition, the surface of MAX1 hydrogels was shown to cause inner and outer membrane disruption in experiments that monitor the release of beta-galactosidase from the cytoplasm of lactose permease-deficient E. coli ML-35. These data suggest a mechanism of antibacterial action that involves membrane disruption that leads to cell death upon cellular contact with the gel surface. Although the hydrogel surface exhibits bactericidal activity, co-culture experiments indicate hydrogel surfaces show selective toxicity to bacterial versus mammalian cells. Additionally, gel surfaces are nonhemolytic toward human erythrocytes, which maintain healthy morphologies when in contact with the surface. These material attributes make MAX1 gels attractive candidates for use in tissue regeneration, even in nonsterile environments.

Light-activated hydrogel formation via the triggered folding and self-assembly of a designed peptide.

Photopolymerization can be used to construct materials with precise temporal and spatial resolution. Applications such as tissue engineering, drug delivery, the fabrication of microfluidic devices and the preparation of high-density cell arrays employ hydrogel materials that are often prepared by this technique. Current photopolymerization strategies used to prepare hydrogels employ photoinitiators, many of which are cytotoxic and require large macromolecular precursors that need to be functionalized with moieties capable of undergoing radical cross-linking reactions. We have developed a simple light-activated hydrogelation system that employs a designed peptide whose ability to self-assemble into hydrogel material is dependent on its intramolecular folded conformational state. An iterative design strategy afforded MAX7CNB, a photocaged peptide that, when dissolved in aqueous medium, remains unfolded and unable to self-assemble; a 2 wt % solution of freely soluble unfolded peptide is stable to ambient light and has the viscosity of water. Irradiation of the solution (260 < lambda < 360 nm) releases the photocage and triggers peptide folding to produce amphiphilic beta-hairpins that self-assemble into viscoelastic hydrogel material. Circular dichroic (CD) spectroscopy supports this folding and self-assembly mechanism, and oscillatory rheology shows that the resulting hydrogel is mechanically rigid (G' = 1000 Pa). Laser scanning confocal microscopy imaging of NIH 3T3 fibroblasts seeded onto the gel indicates that the gel surface is noncytotoxic, conducive to cell adhesion, and allows cell migration. Lastly, thymidine incorporation assays show that cells seeded onto decaged hydrogel proliferate at a rate equivalent to cells seeded onto a tissue culture-treated polystyrene control surface.