Graft Copolymer Stabilized Gold Nanoparticles and Their Applications and Chemically Cleavable Linkers and Their Applications

Abstract

This thesis consists of two parts: (1) graft copolymer stabilized gold nanoparticles (AuNPs) and their biological application and (2) chemically cleavable 𝛼-azido ether and its biological application.

In the first part, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) copolymers that bear multiple thiol groups on the polymer backbone are used for exceptional ligands to stabilize AuNPs. To characterize the effect of copolymer structure on AuNP stability, we synthesized PLL-g-PEGs with different backbone lengths, PEG grafting densities, and number of thiols per polymer chain. AuNPs were then combined with these polymer ligands, and the stabilities of the resulting AuNP@PLL-g-PEG particles against high temperature, oxidants, and competing thiol ligands were characterized using dynamic light scattering (DLS), visible absorption spectroscopy, and fluorescence spectrophotometry. Our observations indicate that thiolated PLL-g-PEG ligands (PLL-g-[PEG:SH]) combine thermodynamic stabilization via multiple Au-S bonds and steric stabilization by PEG grafts, and the best graft copolymer ligands balance these two effects. This new ligand system enables AuNPs to be used for solid phase polymerase chain reaction (SP-PCR) that requires harsh reaction conditions, such as, elevated temperature and competing thiol molecules. Azide functionalized PLL-g-[PEG:SH] were conjugated to oligodeoxy-nucleotide (ODN) primers via click chemistry and bound to AuNPs to yield AuNP-primers that successfully primed target DNA synthesis on the surface of the AuNPs through PCR, as demonstrated by gel electrophoresis, DLS, and fluorescent analysis. Moreover, the graft copolymer stabilized AuNPs were applied to rapid DNA diagnostics in a single PCR tube with magnetic particles through color change without any instrumental analysis.

In the second part, bioorthogonal, chemically cleavable 𝛼-azido ether has been studied and used to develop novel degradable materials. In order to understand the chemistry of the 𝛼-azido ether, model molecules bearing the 𝛼-azido ether were prepared. Hydrolytic stability of the model molecules was investigated by measuring their degradation rate using NMR, which leads to the relationship between the stability and chemical structures. Additionally, the cleavage kinetics of the model molecule, which was triggered by a couple of azide reducing reagents, was studied by NMR and UV-Vis absorption spectroscopy. The kinetic studies enable us to develop mechanistic investigation of the chemical cleavage as well as optimal cleavage conditions. Furthermore, the products after the chemical cleavage of the 𝛼-azido ether were characterized using NMR. The novel 𝛼-azido ether was then incorporated into degradable polyacrylamide gel electrophoresis (PAGE), in which biological macromolecules, including plasmid, microRNA, and proteins, were separated electrophoretically and recovered from the gel matrix with the optimal cleavage conditions. The kinetics of the recovery was quantitatively studied using UV-Vis absorption spectroscopy and fluorescence spectrophotometry. Furthermore, the recovered biological macromolecules were analyzed to investigate biocompatibility of our system. We anticipate further expansion of the 𝛼-azido ether to a broad range of biological applications based on the fundamental studies and the representative example in PAGE.