Tuning the pH responsiveness of beta-hairpin peptide folding, self-assembly, and hydrogel material formation.

A design strategy to control the thermally triggered folding, self-assembly, and subsequent hydrogelation of amphiphilic beta-hairpin peptides in a pH-dependent manner is presented. Point substitutions of the lysine residues of the self-assembling peptide MAX1 were made to alter the net charge of the peptide. In turn, the electrostatic nature of the peptide directly influences the solution pH at which thermally triggered hydrogelation is permitted. CD spectroscopy and oscillatory rheology show that peptides of lower net positive charge are capable of folding and assembling into hydrogel material at lower values of pH at a given temperature. The pH sensitive folding and assembling behavior is not only dependent on the net peptide charge, but also on the exact position of substitution within the peptide sequence. TEM shows that these peptides self-assemble into hydrogels that are composed of well-defined fibrils with nonlaminated morphologies. TEM also indicates that fibril morphology is not influenced by making these sequence changes on the hydrophilic face of the hairpins. Rheology shows that the ultimate mechanical rigidity of these peptide hydrogels is dependent on the rate of folding and self-assembly. Peptides that fold and assemble faster afford more rigid gels. Ultimately, this design strategy yielded a peptide MAX1(K15E) that is capable of undergoing thermally triggered hydrogelation at physiological buffer conditions (pH 7.4, 150 NaCl, 37 degrees C).

Iterative design of peptide-based hydrogels and the effect of network electrostatics on primary chondrocyte behavior.

Iterative peptide design was used to generate two peptide-based hydrogels to study the effect of network electrostatics on primary chondrocyte behavior. MAX8 and HLT2 peptides have formal charge states of +7 and +5 per monomer, respectively. These peptides undergo triggered folding and self-assembly to afford hydrogel networks having similar rheological behavior and local network morphologies, yet different electrostatic character. Each gel can be used to directly encapsulate and syringe-deliver cells. The influence of network electrostatics on cell viability after encapsulation and delivery, extracellular matrix deposition, gene expression, and the bulk mechanical properties of the gel-cell constructs as a function of culture time was assessed. The less electropositive HLT2 gel provides a microenvironment more conducive to chondrocyte encapsulation, delivery, and phenotype maintenance. Cell viability was higher for this gel and although a moderate number of cells dedifferentiated to a fibroblast-like phenotype, many retained their chondrocytic behavior. As a result, gel-cell constructs prepared with HLT2, cultured under static in vitro conditions, contained more GAG and type II collagen resulting in mechanically superior constructs. Chondrocytes delivered in the more electropositive MAX8 gel experienced a greater degree of cell death during encapsulation and delivery and the remaining viable cells were less prone to maintain their phenotype. As a result, MAX8 gel-cell constructs had fewer cells, of which a limited number were capable of laying down cartilage-specific ECM.

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.