Genetically Encoded XTEN-based Hydrogels with Tunable Viscoelasticity and Biodegradability for Injectable Cell Therapies.

While direct cell transplantation holds great promise in treating many debilitating diseases, poor cell survival and engraftment following injection have limited effective clinical translation. Though injectable biomaterials offer protection against membrane-damaging extensional flow and supply a supportive 3D environment in vivo that ultimately improves cell retention and therapeutic costs, most are created from synthetic or naturally harvested polymers that are immunogenic and/or chemically ill-defined. This work presents a shear-thinning and self-healing telechelic recombinant protein-based hydrogel designed around XTEN - a well-expressible, non-immunogenic, and intrinsically disordered polypeptide previously evolved as a genetically encoded alternative to PEGylation to "eXTENd" the in vivo half-life of fused protein therapeutics. By flanking XTEN with self-associating coil domains derived from cartilage oligomeric matrix protein, single-component physically crosslinked hydrogels exhibiting rapid shear thinning and self-healing through homopentameric coiled-coil bundling are formed. Individual and combined point mutations that variably stabilize coil association enables a straightforward method to genetically program material viscoelasticity and biodegradability. Finally, these materials protect and sustain viability of encapsulated human fibroblasts, hepatocytes, embryonic kidney (HEK), and embryonic stem-cell-derived cardiomyocytes (hESC-CMs) through culture, injection, and transcutaneous implantation in mice. These injectable XTEN-based hydrogels show promise for both in vitro cell culture and in vivo cell transplantation applications.

Engineering Antigen-Specific Tolerance to an Artificial Protein Hydrogel.

Artificial protein hydrogels are an emerging class of biomaterials with numerous prospective applications in tissue engineering and regenerative medicine. These materials are likely to be immunogenic due to their frequent incorporation of novel amino acid sequence domains, which often serve a functional role within the material itself. We engineered injectable "self" and "nonself" artificial protein hydrogels, which were predicted to have divergent immune outcomes in vivo on the basis of their primary amino acid sequence. Following implantation in mouse, the nonself gels raised significantly higher antigel antibody titers than the corresponding self gels. Prophylactic administration of a fusion antibody targeting the nonself hydrogel epitopes to DEC-205, an endocytic receptor involved in Treg induction, fully suppressed the elevated antibody titer against the nonself gels. These results suggest that the clinical immune response to artificial protein biomaterials, including those that contain highly antigenic sequence domains, can be tuned through the induction of antigen-specific tolerance.

Catalytic Sulfamoylation of Alcohols with Activated Aryl Sulfamates.

We report a new catalytic method for alcohol sulfamoylation that deploys electron-deficient aryl sulfamates as activated group transfer reagents. The reaction utilizes the simple organic base N-methylimidazole, proceeds under mild conditions, and provides intrinsic selectivity for 1° over 2° alcohols (up to >40:1 for certain nucleosides). The requisite aryl sulfamate donors are stable crystalline solids that can be readily prepared on a large scale. Mechanistic considerations support the intermediacy of HNSO2 "aza-sulfene" in the transfer reaction.

Mechanisms of Diffusion in Associative Polymer Networks: Evidence for Chain Hopping.

Networks assembled by reversible association of telechelic polymers constitute a common class of soft materials. Various mechanisms of chain migration in associative networks have been proposed; yet there remains little quantitative experimental data to discriminate among them. Proposed mechanisms for chain migration include multichain aggregate diffusion as well as single-chain mechanisms such as "walking" and "hopping", wherein diffusion is achieved by either partial ("walking") or complete ("hopping") disengagement of the associated chain segments. Here, we provide evidence that hopping can dominate the effective diffusion of chains in associative networks due to a strong entropic penalty for bridge formation imposed by local network structure; chains become conformationally restricted upon association with two or more spatially separated binding sites. This restriction decreases the effective binding strength of chains with multiple associative domains, thereby increasing the probability that a chain will hop. For telechelic chains this manifests as binding asymmetry, wherein the first association is effectively stronger than the second. We derive a simple thermodynamic model that predicts the fraction of chains that are free to hop as a function of tunable molecular and network properties. A large set of self-diffusivity measurements on a series of model associative polymers finds good agreement with this model.

Analysis and Control of Chain Mobility in Protein Hydrogels.

Coiled-coil domains can direct the assembly of protein block copolymers into physically cross-linked, viscoelastic hydrogels. Here, we describe the use of fluorescence recovery after photobleaching (FRAP) to probe chain mobility in reversible hydrogels assembled from engineered proteins bearing terminal coiled-coil domains. We show that chain mobility can be related to the underlying dynamics of the coiled-coil domains by application of a three-state "hopping" model of chain migration. We further show that genetic programming allows the effective mobility of network chains to be varied 500-fold through modest changes in protein sequence. Destabilization of the coiled-coil domains by site-directed mutagenesis increases the effective diffusivity of probe chains. Conversely, probe mobility is reduced by expanding the hydrophobic surface area of the coiled-coil domains through introduction of the bulky leucine surrogate homoisoleucine. Predictions from the three-state model imply asymmetric sequential binding of the terminal domains. Brownian Dynamics simulations suggest that binding asymmetry is a general feature of reversible gels, arising from a loss in entropy as chains transition to a conformationally restricted bridged state.