Designing multifunctional collagen mimetic peptides to incorporate hierarchical structure within hydrogel biomaterials
Date
2019
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Collagen is one of the most prevalent proteins in the human body, making up approximately 30% of the overall protein content. It exists in ~ 28 different types and contributes to the structure of many tissues such as the skin, bones, and lungs. It assembles through an elegant hierarchal mechanism where three peptide strands come together to form a triple helix, then triple helices interact to form fibrils, and those fibrils further interact to form collagen fibers with widths on the microscale. Collagen can also serve as a cell signaling molecule throughout the body and contains a number of integrin binding sites to allow cells to interact with the protein. Synthetically, shorter peptides called collagen mimetic peptides can be synthesized to mimic this hierarchal assembly process. The goals of this thesis were to design multifunctional collagen mimetic peptides (mfCMPs) that could self-assemble into relevant structures that mimicked native collagen and incorporate a reactive handle within the peptide backbone that could be used independent of the fibrillar assembly process. Additionally, to use the reactive handle to covalently incorporate assembled mfCMPs into a polymer-based hydrogel, characterize the structure property relationships of the resulting materials, then encapsulate cells in three dimensions to study cell response. ☐ First, two different sets of amino acid substitutions were made to the most common collagen triple sequence (POG)n. The first set of substitutions were charged residues lysine and aspartic acids, to promote long-range fibrillar interactions and assembly by ionic interactions. The second substitution was an allyloxycarbonyl protected lysine group which provided an –ene group that could participate in a thiol–ene click chemistry reaction post-assembly. These substitutions were evaluated experimentally and shown to be less stable in solution when compared to the (POG) based sequence based on their melting temperatures likely due to like-charge repulsions on the ends of the sequences. A coarse-grained model was then developed to predict thermal stability trends in proposed peptide designs prior to synthesis using molecular dynamics simulations which showed increased splaying at the ends of the sequences where the charged residues were substituted. Overall the stability trend predicted by the model agreed well with experimental trends. ☐ Second, peptide designs evaluated above were expanded on and characterized on the triple helix and fibrillar scale to study an addition set of substitutions of aromatic stacking groups to promote fibrillar assembly through aromatic interactions. Here, the reactive group was also used to incorporate assembled mfCMPs within hydrogel biomaterials and mechanical properties were shown to remain statistically the same as the control up to a concentration of 2.5 mM peptide. Finally, these hybrid materials were used to encapsulate human mesenchymal stem cells in three dimensions and study their response to a matrix containing assembled structure. At higher mfCMP concentration with the charged sequences we observed a shift in cell morphology from rounded to a more elongated cell morphology, whereas in the case of the aromatic stacking sequences the cells shifted to a more spread morphology. ☐ Lastly, the influence of salt concentration in buffers was examined as a way to tailor stability of an mfCMP on multiple levels of assembly. At the triple helix level increased salt concentration lead to lower melting temperatures indicating less stable triple helices. Fibrillar morphology was impacted based on the measured diameter of observed fibrils. The formation of bulk mfCMP only hydrogels was also studied to determine if they exhibited similar properties to hydrogels made from harvested collagen. When salt was present in solution formation of physical mfCMP hydrogels was observed but at higher concentrations the resulting modulus was ~10x lower than and low salt concentrations. Ultimately mfCMP-PEG hydrogels were robustly formed with varying amounts of mfCMPs used as crosslinks up to 100%. These hydrogels exhibited properties that were reminiscent of both purely elastic gels and physically assembled gels as shown by deviations in the viscoelastic behavior at high strain. Collectively, this thesis has given insight into the ability to tune assembled multifunctional collagen mimetic peptide properties using a variety of handles from amino acid composition, to assembly conditions and processing. The incorporation of a reactive handle that can be used orthogonal to assembly has allowed for the creation of a new nanostructured hydrogel material with tunable properties that can be used to study cell response to assembled structures in vitro.
Description
Keywords
Multifunctional collagen mimetic peptides, Polymer-based hydrogel, Aromatic stacking groups