Rational design and application of responsive alpha-helical peptide hydrogels

Abstract

Biocompatible hydrogels have a wide variety of potential applications in biotechnology and medicine, such as the controlled delivery and release of cells, cosmetics and drugs, and as supports for cell growth and tissue engineering. Rational peptide design and engineering are emerging as promising new routes to such functional biomaterials. Here, we present the first examples of rationally designed and fully characterized self-assembling hydrogels based on standard linear peptides with purely alpha-helical structures, which we call hydrogelating self-assembling fibres (hSAFs). These form spanning networks of alpha-helical fibrils that interact to give self-supporting physical hydrogels of >99% water content. The peptide sequences can be engineered to alter the underlying mechanism of gelation and, consequently, the hydrogel properties. Interestingly, for example, those with hydrogen-bonded networks of fibrils melt on heating, whereas those formed through hydrophobic fibril-fibril interactions strengthen when warmed. The hSAFs are dual-peptide systems that gel only on mixing, which gives tight control over assembly. These properties raise possibilities for using the hSAFs as substrates in cell culture. We have tested this in comparison with the widely used Matrigel substrate, and demonstrate that, like Matrigel, hSAFs support both growth and differentiation of rat adrenal pheochromocytoma cells for sustained periods in culture.

Figure 1. hSAF design principles

(a) In previous SAF designs, specific charged interactions between certain b and c positions lead to peptide alignment and fiber thickening. (b) For the hSAFs, we replaced these specific interactions with weaker, more-general interactions at all b, c and f sites, to result in smaller, more flexible, bundles of thinner fibres.

Figure 2. Gel strength and network formation by the hSAFs

(a) Gel strength gauged by nondestructive oscillatory rheology. Key: G’ (solid symbols) and G” (open); hSAF_AAA (discs)and hSAF_QQQ (squares). Measurements were made at a strain of 0.5 %. (b - e) Network formation as observed by cryoSEM for: hSAF_QQQ (a), hSAF_AAA (b), and hSAF_AAQ (c), all assembled on ice for 15 minutes; and (d) for hSAF_AAA assembled on ice for 3 minutes and then at room temperature for 12 minutes. All images are at the same magnification.

Figure 3. α-helical secondary structure and packing within the fibrils and gels

CD spectra (a and c) and x-ray fiber diffraction patterns (b and d) for hSAF_AAA (a and b) and hSAF_QQQ (b and d).

Figure 4. Cell growth and differentiation on hSAF hydrogels

Phase-contrast microscopy of differentiating rat adrenal pheochromocytoma (PC12) cells in hSAF_AAA-W hydrogel (a), and matrigel (b). These images were taken 10 days after adding nerve growth factor. Images from the full 14-day time course are given in the Supplementary information. Cell differentiation as observed by neurite outgrowth was semi-quantified over time by: (c) the lengths of the processes; and (d) the percentage of cells showing processes. In both plots, error bars show the standard error of the mean for measurements from ≥ 100 cells/cell clusters across 10 different fields of view in three different triplicate wells.