The Design, Characterization, and Utilization of Self-Assembling Biomaterials for Diagnostic and Therapeutic Applications

Abstract

Self-assembling biomaterials are biologically mimicking, or otherwise biologically interactive, materials. Their bottom-up organization has been exploited for various biomedical applications, including therapeutic delivery, biomedical imaging, and dual therapeutic delivery and diagnostics, or theranostics. Inspired by complex systems in nature, biomaterials have been engineered using both synthetic and biosynthesized monomers. While synthetic materials, particularly synthetic phospholipids and polymers, have been widely described and included in FDA-approved therapies, advances in protein engineering have led to increased studies taking advantage of their genetically-designed size, structure, and functionality. The work of this thesis aimed to design, synthesize, and characterize three distinct self-assembling biomaterials, as well as explore their biomedical applications, with each at a different stage in development.

First, by taking advantage of commercially available synthetic phospholipids, a phospholipid-based gadolinium-bearing micelle was synthesized. Its diagnostic potential was explored as a blood pool contrast agent for magnetic resonance angiography (MRA) of the C57BL/6 wild type murine cerebrovasculature over two years of aging. Micelle enhanced-MRA detected a significant loss of vasculature, or rarefaction, during aging, which was confirmed via immunohistochemistry. While vascular rarefaction has been described in human and rat subjects, it has rarely been discussed in murine models; its detection in this work suggests that age-dependent cerebrovascular changes must be investigated, and accounted for, in murine models of age-dependent cerebrovascular diseases.

Second, transitioning from synthetic to biosynthesized materials, a nanofiber-assembling engineered coiled-coil protein, dubbed Q, was explored for its use in hydrogel assembly. Q formed a physically crosslinked hydrogel demonstrating nanofiber entanglement, which was capable of binding the small molecule curcumin, a model drug for hydrophobic chemotherapeutics. Curcumin binding increased mechanical integrity and thermostability, enabling sustained drug release from the hydrogel at physiological temperature.

Third, by altering buffer conditions, Q assembled into nanofibers with diameters 10x larger that lacked entanglement and, following doxorubicin binding, further assembled into mesofibers. Using non-natural amino acid incorporation, an azide-functionalized Q protein was conjugated to an alkyne-bearing iron oxide templating peptide. The construct improved the therapeutic effect of doxorubicin in vitro and organized ultrasmall superparamagnetic iron oxide nanoparticles to generate a protein fiber-iron oxide hybrid material detectable by its enhanced darkening effect on MRI, providing potential as a theranostic agent for MRI-traceable drug delivery.

Overall, this work explored both traditional and newly described techniques in engineering self-assembling biomaterials for diagnostic, therapeutic, or theranostic applications. These three unique materials have potential for visualizing age-dependent cerebrovascular changes that may confound studies of tumors, stroke, and Alzheimer’s disease; demonstrating sustained release of small molecules such as chemotherapeutics; and providing MRI-traceable chemotherapeutic delivery, respectively.