Graduation Year

2016

Document Type

Thesis

Degree

M.S.B.E.

Degree Name

MS in Biomedical Engineering (M.S.B.E.)

Degree Granting Department

Chemical Engineering

Major Professor

Piyush Koria, Ph.D.

Committee Member

Mark Jaroszeski, Ph.D.

Committee Member

Nathan Gallant, Ph.D.

Keywords

Biomaterials, recombinant proteins, epidermal growth factor, protein polymers, photocrosslinking

Abstract

Elastin like polypeptides (ELPs) are a class of naturally derived biomaterials that are non-immunogenic, genetically encodable, and biocompatible making them ideal for a variety of biomedical applications, ranging from drug delivery to tissue engineering. Also, ELPs undergo temperature-mediated inverse phase transitioning, which allows them to be purified in a relatively simple manner from bacterial expression hosts. Being able to genetically encode ELPs allows for the incorporation of bioactive peptides and functionalization of ELPs. This work utilizes ELPs for regenerative medicine and drug delivery.

The goal of the first study was to synthesize a biologically active epidermal growth factor-ELP (EGF-ELP) fusion protein that could aid in the treatment of chronic wounds. EGF plays a crucial role in wound healing by inducing epithelial cell proliferation and migration, and fibroblast proliferation. The use of exogenous EGF has seen success in the treatment of acute wounds, but has seen relatively minimal success in chronic wounds because the method of delivery does not protect exogenous EGF from degradation, or prevent it from diffusing away from the application site.

We created an EGF-ELP fusion protein to combat these issues. As demonstrated through the proliferation of human skin fibroblasts in vitro, the EGF-ELP may be able to aid in the treatment of chronic wounds. Furthermore, the ability of the EGF-ELP to self-assemble near physiological temperatures could allow for the formation of drug depots at the wound site and minimize diffusion, increasing the bioavailability of EGF and enhancing tissue regeneration.

The objective of the second study was to create an injectable hydrogel platform that does not require conjugation of functional moieties for crosslinking or biological activity. Hydrogels are three-dimensional polymer networks that are able to absorb water and biological fluids without dissolving. Their high water content gives them physical properties similar to soft tissues, making them useful as scaffolds for cell migration and drug delivery vehicles. Injectable hydrogels that crosslink in situ are particularly useful because they can form to the shape of the defect, providing a near perfect fit. However, many hydrogel platforms cannot be crosslinked in situ because cytotoxic crosslinking reagents are required. Additionally, hydrogels typically require the chemical conjugation of crosslinking domains and bioactive peptides to the polymer backbone, adding more steps and time required for hydrogel production.

We devised an injectable hydrogel platform that can be synthesized in a single step using photoreactive ELPs as the polymer backbone. Leucine auxotrophic Eshcherichia coli expressed ELPs containing photoleucine, a leucine analog and photoreactive diazirine crosslinker, which is substituted for leucine periodically throughout the ELP sequence. Upon exposure to ultraviolet radiation (~370 nm), photoleucine is able to form covalent crosslinks with amino acid side chains, forming a polymer network for hydrogel formation. Additionally, recombinant growth factors and morphogens can be encoded into the ELP sequence providing a simple method of hydrogel functionalization for regenerative medicine applications.

The potential for this platform was demonstrated through in vivo crosslinking of photoreactive ELPs in the expression hosts. Though the production of the photoreactive ELP was not as forthright as originally assumed. The substitution of noncanonical amino acids typically requires the auxotrophic expression hosts to be starved of the amino acid that they are auxotrophic for. A noncanonical analog of said amino acid can then be supplemented into expression media, maximizing incorporation. In this investigation, it was found the addition of photoleucine alone inhibited photoreactive ELP expression. ELP expression only occurred in the presence of photoleucine if valine or leucine was also present in the media. Furthermore, valine was found to aid the production of ELPs as much as leucine. It was postulated the bacterial translational machinery might need to be altered for optimal ELP expression.

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