(531a) Bio-Orthogonally Crosslinked, Engineered Protein Hydrogels with Tunable Mechanics and Biochemistry for Cell Encapsulation

Covalently crosslinked hydrogels are commonly used as 3D materials to study cell-matrix interactions and for therapeutic delivery of cells in regenerative medicine applications. While covalent crosslinking provides increased material stability and enhanced mechanical rigidity, most of the crosslinking chemistries also react with chemical moieties present on encapsulated cells and biomolecules. Recent progress in the development of bio-orthogonal reactions that eliminate these side reactions can be employed to produce hydrogels with highly specific crosslinking. However, previous hydrogel systems prepared using some of these chemistries are limited by a variety of factors, including copper cytotoxicity, non-ideal gelation kinetics, batch-to-batch variability, and the inability to decouple mechanical properties, cell adhesivity, and degradation. In response, we have developed an engineered protein hydrogel crosslinked via strain-promoted azide-alkyne cycloaddition (SPAAC) that gels in seconds, preserves cell viability and function, permits independent tuning of cell-adhesive ligand concentration and hydrogel stiffness, and allows for selective chemical functionalization of the gels in the presence of living cells and serum. Engineered elastin-like proteins (ELPs) containing either a cell-adhesive RGD domain derived from human fibronectin or a non-adhesive, scrambled RDG domain were functionalized with azides and bicyclononynes (BCN) to facilitate SPAAC crosslinking. To form a gel, ELP modified with azides is simply mixed with ELP modified with BCN. SPAAC crosslinking occurs within seconds, and gelation is complete within minutes. Rapid gelation kinetics facilitate uniform cell dispersion within the hydrogels and are desirable for applications such as cell transplantation through injection and bioprinting, in which fast gelation may increase material retention at the site of injection and improve the fidelity of 3D-printed structures. To emphasize the importance of choosing a chemistry with such rapid gelation kinetics, ELPs were also modified with triarylphosphines to permit crosslinking via the bio-orthogonal Staudinger ligation reaction. Staudinger-crosslinked ELPs gelled in tens of minutes, a timescale too slow for successful cell encapsulation. By varying the extent of BCN modification and the total protein content in the rapidly gelling SPAAC-crosslinked ELPs, the storage modulus (Gâ??) of the material was varied from 180 Pa to 3300 Pa. Furthermore, at a fixed total polymer content, varying the extent of BCN modification and the ratio of ELP containing cell-adhesive RGD peptides vs. non-adhesive RDG peptides permitted independent tuning of Gâ?? (from ~1200 to ~3300 Pa) and RGD concentration (from 0 to 5.3 mM). The cytocompatibility of the crosslinking chemistry was assessed by live/dead staining of encapsulated mesenchymal stem cells (MSCs), endothelial cells (ECs), and neural progenitor cells (NPCs) one hour post-gelation. All three cell types remained highly viable (97-99%). Furthermore, the SPAAC-crosslinked ELPs permitted phenotypic maintenance of the cells in culture, with ECs forming tubular networks with open lumen-like structures and NPCs retaining the capacity to differentiate into neurons and astrocytes. Additionally, the importance of decoupled mechanics and adhesive ligand concentration was demonstrated using MSCs. MSCs in 5.3 mM RGD gels exhibited well-spread morphologies, while cells in 0 mM RGD gels remained rounded, independent of hydrogel stiffness. However, in gels with an intermediate RGD density (0.53 mM), significant spreading was only observed in 1200 Pa gels, not in 3300 Pa gels. Finally, the bio-orthogonality of the SPAAC crosslinking was demonstrated by functionalizing excess azide residues in MSC-laden gels with a BCN-bearing fluorescent probe in serum-containing culture medium. Only gels treated with the BCN-containing probe, and not a control, amine-containing probe, displayed significant fluorescence. These results demonstrate that ELP hydrogels crosslinked via SPAAC facilitate independent tuning of matrix stiffness and ligand chemistry, and SPAAC-ELPs permit efficient cell encapsulation and phenotypic maintenance in vitro. The bio-orthogonal nature of the crosslinking chemistry makes SPAAC-crosslinked ELP hydrogels attractive materials for applications such as cell delivery and bioprinting, while also permitting highly selective functionalization of the material in the presence of living cells.