Date of Award

8-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Materials Science and Engineering

Committee Member

Dr. Stephen H. Foulger, Committee Chair

Committee Member

Dr. Igor A. Luzinov

Committee Member

Dr. O. Thompson Mefford

Committee Member

Dr. Michael G. Sehorn

Abstract

The main objective of this project is to develop a polymer based near infrared (NIR) particle that can serve as both, diagnostic and therapeutic agents, for fighting cancer. Specifically these particles will be designed to have high-contrast, high signal to noise ratios, long in vivo circulation lifetimes, and facilitate easy attachment of functional and target components. Cancer is a disease where the growth of abnormal cells is uncontrollable and is one of the leading causes of death worldwide. Malignant cancer cells are more effectively treated when identified early in the disease. Identification of tumors using fluorophores, small molecules that emit visible light when excited, is gaining clinical interest. Specific interest is in near-infrared emission due to the lack of absorption of this radiation in human tissue, which facilitates deep tissue imaging. During imaging with small molecule fluorophores, the fluorophores clear from the body quickly reducing imaging effectiveness. The effectiveness of imaging can be enhanced by attaching the fluorophores to a particle. Nanometer sized particles do not clear from the body rapidly and allows the material designer to attach other ”payloads” to the particle. This multifunctional ”nano-device” can be used to deliver diagnostic (fluorophores) and therapeutic (drugs) agents to the afflicted tissue. In this current project sub-100 nm poly(propargyl acrylate) (PA) particles are surface-functionalized with fluorophores or targeting molecules through a copper(I) catalyzed azide-alkyne Huisgen 1,3-Dipolar cycloaddition, and used for the following applications: (1) Switching fluorescence of surface modified colloids with near-infrared emitters via pro-tein interaction for contrast-enhanced imaging: The colloidal particles surface-functionalized with fluorophores exhibit a protein triggered activation/deactivation of the emission. Dispersing the par-ticles into an aqueous solution, such as phosphate buffered saline (PBS), results in an aggregation of the hydrophobic fluorophores and a cessation of emission. The emission can be reinstated, or activated, by the conversion of the surface-attached fluorophores from an aggregate to a monomeric species with the addition of an albumin. This activated probe can be deactivated and returned to a quenched state by a simple tryptic digestion of the albumin. The methodology for emission switching offers a path to maximize the signal from the typically weak quantum yield inherent in NIR fluo-rophores. Preliminary fluorescence imaging studies indicate that the brightness of the functionalized polymer based nanoparticles improved considerably. (2) Surface modified colloids with targeting molecules to disrupt Survivin activity and en-hance apoptosis in cancer cells: Survivin belongs to the family of inhibitor of apoptosis proteins (IAP) and is present in most cancers while being below detection limits in most terminally differentiated adult tissues, making it an attractive protein to target for diagnostic and, potentially, therapeutic roles. Sub-100 nm poly(propargyl acrylate) (PA) particles which are surface-functionalized with an azide terminated Survivin ligand derivative (azTM), originally proposed by Abbott Labs and speculated to bind directly to Survivin (protein) at its dimer interface. Using affinity pull-down studies, it was determined that the PA/azTM nanoparticles selectively bind Survivin and the parti-cles can enhance apoptotic cell death in glioblastomas and other Survivin over-expressing cell lines such as A549 and MCF7 relative to cells incubated with the original Abbott-derived small molecule inhibitor. (3) A bioresponsive and versatile particle-protein-dye system for small molecule delivery and FRET based imaging: Colloidal particles are surface-functionalized with small molecules such as fluorophores and ligands through an environmentally-sensitive linker. The linker used is an azide modified bovine serum albumin (azBSA) which prevents opsonization and releases the small molecule upon digestion. Attachment of the fluorophore to the particle through the azide modified bovine serum albumin (BSA) quenches the emission and is specifically activated upon the denature or digestion of the azBSA. This resulted in an enhanced tumor to background signal ratio. A fluorescence resonance energy transfer (FRET) pair of dyes can be attached to the particle and the azBSA, and the FRET efficiency can be increased by unfolding the protein. These dye modified particles gave promising results in photodynamic therapy studies performed in human head and neck squamous carcinoma cells (UMSCC22A). Further, a targeting ligand can also be attached to the particles using the same strategy to achieve higher accumulations of the desired small molecule inside the tumor. In addition, preliminary imaging and toxicity studies were carried out in human lung carcinoma cells (A549) using a Survivin targeting ligand attached to PA-azBSA particles and an enhanced cell death relative to free molecule treatment was observed.

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