| dc.description.abstract | According to the American Cancer Society, approximately 1,479,000 new cases of cancer were expected to be diagnosed, while 562,340 Americans were expected to die from cancer in 2009 alone. Even though advances in early diagnosis and therapy over the past few decades have led to continual decreases in incidence and mortality, cancer remains the second leading cause of death among all Americans. Consequently, further technological development in all areas of cancer detection and treatment are still of great importance not only to the scientific community, but to society itself. To address the shortcomings in current cancer diagnosis and treatment, a novel, highly adaptable, targeted nanoparticle system based on alternating amphiphilic copolymers has been developed having a variety of potential clinical applications. These polymers consist of an alternating copolymer backbone composed of hydrophilic polyethylene glycol-900 (PEG900) and dimethyl 5-hydroxyisophthalate (linker) monomer units. The linker within the backbone polymer has a free hydroxyl group to which a variety of sidechains can be attached, including hydrophobic groups to impart amphiphilicity, targeting ligands, as well as contrast agents for imaging applications. Three major areas of investigation were addressed to develop and evaluate the performance of the proposed amphiphilic alternating copolymers: (1) backbone polymer synthesis, (2) attachment of radioiodine, and (3) targeted delivery in vitro and in vivo. The first step in the production of the alternating amphiphilic copolymers is a chemo-enzymatic condensation polymerization of polyethylene glycol (PEG) and dimethyl 5-hydroxyisophthalate (linker) to produce backbone polymer. Because of their generally low equilibrium constants, condensation polymerizations require effective removal of the condensation byproduct (in this case, methanol) in order to achieve significant increases in molecular weight. The increased viscosities at higher molecular weights not only increase the difficulty of byproduct removal, but may also affect the mixing characteristics as well as the mass transfer of other species in the reaction. The enzymatic polymerization was investigated using both predictive modeling and experiment. The ultimate goal was to increase the molecular weight of the synthesized polymer to allow for increased substitution of the polymer backbone. Key experimental variables were tested in glass flasks typically used in organic synthesis. In these reactions, 4A molecular sieves had the greatest affect on the backbone polymer molecular weight. In particular, addition of sieves, which can act as sinks for both water and methanol, led to a twofold increase in weight-average molecular weight above that observed previously for the enzymatic polymerization. The Protherm, a novel, thin-film reactor was employed in order to improve methanol mass transfer and mixing within the polymer melt. Three separate reactions in the Protherm produced the highest Mw backbone polymer (approximately 20 kDa). A blade speed of 500 rpm with molecular sieves present was able to achieve this Mw in 48 hr. Two separate models were proposed to describe the polymerization, including a homogeneous kinetic model and a Fick's Law mass transfer model. Significant differences were observed between the experimental results and the predictions of the homogeneous model. The mass transfer modeling, which estimated the increase in reactant and methanol surface concentration relative to the concentration in the bulk, was unable to bridge the gap between experiment and model results. Limited knowledge of key model parameters, including the equilibrium constant and methanol solubility, was one proposed explanation for the observed discrepancy. In order to assess the performance of a nanoparticle delivery system in biological applications, a label that is detectable under a wide range of conditions and concentrations must be present within the molecule. Radioiodine was selected because of its multiple potential applications depending on the selected isotope, including 124I for positron emission tomography, 131I for radiotherapy, and 125I for inexpensive, quantitative research applications. A standard protein-labeling technique was adapted for application to the copolymers in this work. The successful adaptation of this procedure for use with our polymers represented the first demonstration in the field of a nanoparticle-forming polymer that was directly labeled with radioiodine without any additional chemicalalterations or intermediate reactions. The process was characterized using a variety of chromatographic techniques and radiometric measurements to confirmed covalent, stable attachment of iodine in a product with high radiochemical purity. The alternating amphiphilic copolymers were combined with an engineered peptide having an extremely high binding affinity for the epidermal growth factor receptor (EGFR), a biomarker prevalent in a variety of human cancers. This high-affinity binder, the E13.4.3 peptide, was developed by collaborator Dr. Benjamin Hackel under the guidance of Professor K. Dane Wittrup. A number of polymer design variables were considered, including the targeting ligand density, identity of the hydrophobic sidechain, polymer molecular weight, and length of the spacer connecting the peptide to the backbone. The ligand density and hydrophobic sidechain identity were chosen for study. Initial studies demonstrated selective uptake of E13.4.3-conjugated polymers into a target-bearing, EGFR-positive human cancer cell line relative to untargeted controls. Preparative gel permeation chromatography (GPC) was used to create high molecular weight, low polydispersity fractions of backbone polymer. Polymers synthesized from these fractions achieved the greatest increase in selective uptake in vitro with a four- to sixfold increase in uptake for E13.4.3-conjugated polymers relative to untargeted controls. Animal studies measured the biodistribution, blood circulation, and tumoral accumulation of various polymer formulations. Statistically significant selective tumor accumulation was observed for two different targeted polymers, each having different targeting ligand density and different hydrophobic sidechains. The E13.4.3-polymers have proven a rich platform for study. Their demonstrated ability to selectively accumulate in targeted tumors combined with their potential use in diagnostic and/or therapeutic clinical applications makes them an attractive option for intensified investigation. | en_US |
