Elastin-like protein hydrogels : a synthetic material platform for neural and cardiac therapeutics

From a Tissue Engineering standpoint, material properties are used to regulate cellular responses. In this pursuit, the field has looked extensively at understanding the influence of the extracellular matrix (ECM) and endogenous cells to draw inspiration for materials design. This study, however, presents a significant challenge to the materials engineer. First, the ECM has numerous properties such as matrix stiffness, stress-relaxation, adhesive ligand concentration and others that vary from tissue to tissue. Additionally, it is known that each of these properties can independently, and in tandem, influence cell response both in vitro and in vivo on a cell-dependent level. Though a challenge, the knowledge gained from this understanding would pave the way for designing therapeutics to meet the needs of numerous clinical needs. However, this requires the design of new materials that allow for such an exact and reproducible degree of control to allow for systematic study. Biomaterials can be broken down into two broad categories: synthetic and naturally derived. Naturally derived materials have the benefit of being biologically active, having relevant matrix stiffness ranges and being readily biodegradable but are limited by their inherent variability due their sourcing. Conversely, synthetic materials are well-defined and have reproducible material properties but lack native bioactivity. Recombinant protein engineering offers a unique toolset that can bridge these two systems by allowing for the design of synthetic DNA that can be used to exactly control the identity of a target protein. By extension, this affords the engineer direct control over the bioactivity and potential crosslinking schemes that can influence the resulting matrix mechanics. The work in this thesis makes extensive use of a class of engineered materials known as elastin-like proteins (ELP). These ELP's are comprised of repeating units of bioactive regions and elastic-like regions. Broadly, the bioactive region comprises of amino acid sequences that allow for cell-matrix interactions through ligand and cell-receptor engagement. The elastin-like region is a sequence of amino acids based off the human-elastin VPGXG pentamer repeat, where the 'X' amino acid is modified at regular intervals to contain chemically active (e.g., lysine or tyrosine groups) amino acid moieties to allow crosslinking of the material. Importantly, the functionality of these two regions is decoupled to allow for independent tuning of the bioactivity and matrix mechanics. Throughout this work, both properties are leveraged in vivo for pre-clinical proof-of-concept studies and the potential applicability of the system is expanded through the engineering of new ELP's with various ECM-inspired proteins. The first part of this work (Chapters 2 and 3) is focused primarily on proof-of-concept translational studies where ELP-based hydrogels were tuned for the bioactivity and mechanical properties for two distinct applications. In Chapter 2, the therapeutic potential of fibronectin-derived RGD-containing ELP's as nerve guidance channels were studied in a 6-week full transection sciatic nerve injury model was explored. Our results indicated that compared to empty conduits (sans luminal filler), animals which received an ELP-filled conduit had both significantly higher functional control and significantly higher degree of functional recovery. Additionally, it was found that they had overall higher amplitude and conduction velocity, but the differences were not significant. In Chapter 3, hyaluronan (HA) and elastin like protein (HELP) gels were formed by combining either benzaldehyde or aldehyde modified HA and hydrazine functionalized ELP. These gels have the same independent tunability (matrix mechanics and bioactivity) as standard ELP-hydrogels but have the added benefit of a stress-relaxation property. In this study, we demonstrated that the relative injectability of HELP hydrogels is tied to failure stress of the material, and that this failure stress can be tuned by modulating the chemical modification scheme of the HA specifically. Leveraging this injectability we further demonstrate that HELP gels significantly improve retention of a fluorescent cargo compared to commercially available Matrigel when injected directly into the myocardium both 24 hours and 7 days post injection. Combined, these projects demonstrate how either the bioactivity or matrix mechanics of ELP can be tuned per the needs of a given application The last part of this work focuses on expanding the versatility of our ELP hydrogel platform by engineering new ELP's with cell-instructive ligands from laminins, collagen, and tenascin-C. Through this project, new ELP are designed, cloned, expressed, and further validated through western blotting, amino acid analysis, and NMR. It is further shown that the lower critical solution temperature (LCST) property of the ELP is directly influenced by the functionalization, where hydrophobic side groups (e.g., azide, BCN groups) suppress the LCST whereas hydrophilic moieties (e.g., hydrazine) raise the LCST. Ultimately, leveraging HELP hydrogel chemistry it is shown that RGD-, YIGSR-, DGEA-, PLAEIDGIELTY-, VFDNFVL-, and RDG-ELP can form hydrogels that have the same independently tunable bioactivity and matrix mechanics but now with an extended selection of ligands making the system more amenable to systematic tuning. As a proof of concept, it is shown that embryonic rat dorsal root ganglion can be cultured in single and dual ligand hydrogels and that the degree of neurite outgrowth varies with the ligand type and formulation. Taken together the work presented in this thesis, principally, demonstrates that versatility and range of tunability for ELP hydrogels for both in vitro and in vivo applications. This lays the groundwork for long-term in vivo studies for both peripheral nerve injury and cardiac therapies where current clinical options have significant drawbacks and limitations. Lastly, by expanding the numbers of ELP it further makes this material system more encompassing by allowing the systematic study of cell processes in a 3D context with an all-in-one, controlled, synthetic platform

Keywords: Materials Science and Engineering.
Advisor: Heilshorn Sarah
Appel Eric (Eric Andrew)
George Paul M. (Paul Matthew)