Two-component protein-engineered physical hydrogels for cell encapsulation

Abstract

Current protocols to encapsulate cells within physical hydrogels require substantial changes in environmental conditions (pH, temperature, or ionic strength) to initiate gelation. These conditions can be detrimental to cells and are often difficult to reproduce, therefore complicating their use in clinical settings. We report the development of a two-component, molecular-recognition gelation strategy that enables cell encapsulation without environmental triggers. Instead, the two components, which contain multiple repeats of WW and proline-rich peptide domains, undergo a sol-gel phase transition upon simple mixing and hetero-assembly of the peptide domains. We term these materials mixing-induced, two-component hydrogels. Our results demonstrate use of the WW and proline-rich domains in protein-engineered materials and expand the library of peptides successfully designed into engineered proteins. Because both of these association domains are normally found intracellularly, their molecular recognition is not disrupted by the presence of additional biomolecules in the extracellular milieu, thereby enabling reproducible encapsulation of multiple cell types, including PC-12 neuronal-like cells, human umbilical vein endothelial cells, and murine adult neural stem cells. Precise variations in the molecular-level design of the two components including (i) the frequency of repeated association domains per chain and (ii) the association energy between domains enable tailoring of the hydrogel viscoelasticity to achieve plateau shear moduli ranging from approximately 9 to 50 Pa. Because of the transient physical crosslinks that form between association domains, these hydrogels are shear-thinning, injectable, and self-healing. Neural stem cells encapsulated in the hydrogels form stable three-dimensional cultures that continue to self-renew, differentiate, and sprout extended neurites.

Fig. 1.

Schematic of the mixing-induced, two-component hydrogel. (Top left) Modular association domains assemble via molecular recognition. Two WW domains (CC43 and a Nedd4.3 variant) bind the same proline peptide (PPxY). (Bottom left) Hydrophilic spacers link multiple repeats of WW domains (spacer 1) or proline peptides (spacer 2). Spacer 1 contains a cell-adhesion peptide RGDS. (Right) Three engineered protein families: C[x + 2], N[y + 2], and P[z + 2]. Mixing component 1 with component 2 (either C[x + 2] or N[y + 2]) at constant physiological conditions results in hydrogel formation.

Fig. 2.

Microrheological characterization. (A) Mean-squared displacement (MSD) of fluorospheres embedded within solutions of C3, C7, N3, N7, P3, and P9 (7.5 wt %). Dashed line indicates MSD power-law scaling of τ¹. (B) Mean-squared displacement of mixed solutions of C3:P3, C7:P3, C3:P9, and C7:P9 (7.5 wt %). (C) Mean-squared displacement of mixed solutions of C7:P9 and N7:P9 (7.5 wt %).

Fig. 3.

Hydrogel shear-thinning and self-healing. Mean-squared displacement (MSD) of (A) C7:P9 and (B) N7:P9 gels (7.5 wt %) before and at multiple times after shear-thinning through a syringe needle.

Fig. 4.

Encapsulated human umbilical vein endothelial cells. (A) Viewing orientation relative to the cell–gel mixture. (B) Confocal z-plane micrographs of viable (green, calcein AM) and dead (red, ethidium homodimer) cells, indicating dispersion throughout C7:P9 and N7:P9 gels (7.5 wt %). (Scale bars, 50 μm.)

Fig. 5.

Encapsulated adult neural stem cell differentiation. Confocal z-stack projections of NSCs differentiated within C7:P9 and N7:P9 gels (5 wt %) at 6 days (red, glial marker GFAP; green, neuronal marker MAP2; yellow, progenitor marker nestin; blue, nuclei, DAPI). (Scale bars, 25 μm.)