Tuning Hydrogel Properties Thorough Simple Sequence Modification of Genetically Encoded XTEN-based Hydrogels

Introduction:: Cellular therapies provide great promise for improved treatment of a vast number of diseases, however the direct injection of cell often results in significant cell death and low cell engraftment. As such, cellular therapies remain difficult to translate to the clinic due to barriers in cost and efficiency. Injectable biomaterials hold promise in improving cellular delivery by providing cells with protection from harsh injection force and a supportive environment post-injection, thereby lowering the number of cells needed for effective engraftment.¹ However, previously developed hydrogels intended for cell delivery have either utilized synthetic polymers, which have raised concerns in their immunogenicity and biodegradability, or tissue-derived biomolecules that lack tunability and often suffer from batch-to-batch variability.² Instead, we proposed the use of recombinant protein biomaterials, which provide significant tunability through modification of the primary sequence, intrinsic biodegradability, and have minimal concern for immunogenicity. Towards this end, we created a telechelic protein centered on XTEN, a polypeptide previously designed to extend the half-life of therapeutics in vivo, and flanked with spontaneously self-assembling coil sequences.^3,4 This single protein, denoted PXP, is able to form macroscopic gels through coiled-coil crosslinking. We strategically mutated residues within these coils to modify gel mechanics and degradation rates, and validated the cytocompatibility of and the injection protection afforded by the materials for both fibroblasts and cardiomyocytes.

Materials and Methods:: PXP and its sister mutants Q54A, T40A, and Q54A+T40A were produced through expression in E. coli followed by Ni-NTA affinity chromatography with endotoxin removal. Final pure proteins were dialyzed into water, sterile filtered, and lyophilized. Lyophilized powder was easily resuspended at 10% (w/v) in PBS to yield a hydrogel. PXP and all mutants were mechanically characterized by oscillatory rheology to determine the storage modulus, frequency and strain crossovers, shear thinning capabilities, and self-healing behavior. Gel erosion studies were carried out in excess PBS over the course of 30 days and protein content was used to determine percent degradation. Finally, both 3T3 fibroblasts and human embryonic stem cell derived cardiomyocytes were separately encapsulated in each gel type and cell viability was assessed through injection and culture up to 72 hours.

Results, Conclusions, and Discussions:: We find that the ability to strategically modify residues within the coils allows for direct modulation of the storage moduli, strain, and frequency crossovers. Mutant gels resulted in lower strain crossovers and extended linear viscoelastic ranges. However, due to the non-covalent nature of all four networks, they all retain clear shear thinning and self-healing behaviors. Additionally, these modifications to the coil sequences change the rate of gel degradation, which follows a surface erosion profile. All mutant gels show slower degradation of the gel, with erosion plateauing after 10-12 days rather than 4 days for PXP. Finally, we show that encapsulation in any of these gels increases cell viability after injection when compared to injection as a liquid suspension. This increase is smaller for fibroblasts, which are known to be less sensitive cell type, whereas the increase in survival of cardiomyocytes when protected by our hydrogel network is >30%.

This work details a set of recombinant protein hydrogels whose mechanical properties and degradation profiles can be varied by single point mutations. The changes resulting from these small mutations demonstrate the important contributions of protein primary sequence to final gel properties and allows us to imagine just how broad the exploration space of engineered protein materials could be. This material is also the first to utilize the uniquely evolved XTEN polypeptide into its structure, adding XTEN to the arsenal of unstructured protein linkers available for incorporation into protein biomaterials. Furthermore, we corroborate the importance of hydrogels as an essential component for cellular delivery via injection and demonstrate that this new class of recombinant protein hydrogels is well-suited for the application.

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References (Optional): :
1. B. A. Aguado Tissue Engineering. Part A, 2012; 18, 806.




2. Tibbitt MW. Biotechnol. Bioeng. 2009; 103:655–663.




3. Petka WA. Science, 1998; 281:389–392.




4. Schellenberger V. Nat. Biotech. 2009;27:1186–1190.