Over the past few decades, tremendous efforts have been made to develop nanomaterials for biotechnological applications such as therapeutics. Understanding and engineering interfaces between biomacromolecules and nanomaterials is a key to the creation of successful therapeutic systems. My research has been oriented toward developing therapeutic systems using gold nanoparticles (AuNPs) incorporating material science, organic synthesis, and biology. For this purpose, mixed monolayer protected AuNPs (~2 nm core size) with various functional groups have been employed for triggering therapeutic effects. Several strategies have been accomplished using anticancer drugs that non-covalently and covalently incorporate onto AuNPs as a drug delivery carrier. Alternatively, AuNPs were developed by regulating host-guest complexation processes inside the cell, allowing control of the therapeutic effect of the AuNP. In addition, by using host-guest chemical events on the AuNPs, exocytosis of the AuNPs was controlled, enabling their prolonged retention inside of the cells, providing new strategies for improving conventional drug delivery systems. Therefore, engineering of the AuNP surface can afford new pathways for designing and improving therapeutics.

<p>This thesis has exploited the use of gold nanoparticles (AuNPs)/DNA conjugates towards 1) the development of simple colorimetric assays to monitor DNA functions and relevant biological processes, and 2) the control the nanoassembly of AuNPs using biomolecules and biological processes.</p> <p>DNA has a number of attractive functions including specific biorecognition, catalysis and being manipulated by protein enzymes, etc. These characteristics were exploited to permit nanoassembly to be responsive to a specific stimulus and also ensure the specificity and precision in the construction of well-defined 3D nanostructures. Meanwhile, the assembly or disassembly of AuNPs, which results in distinct color changes due to the localized surface plasmon resonance, provides an excellent platform for the colorimetrically monitoring the DNA functions and the relevant biological processes.</p> <p>We have specifically investigated how the surface charges, the length and conformations of surface-tethered DNA polymers affect the assembly of AuNPs. We found that the colloidal stability of AuNPs can be well-tuned by nucleotides (small charged molecules) with various binding affinity to AuNP surface and/or different number of negatively-charged phosphate groups. This relies on the fact that nucleotides can bind to AuNP surface via nucleobase-Au interaction, and negatively charged phosphates stabilize AuNPs via electrostatic repulsion. This investigation allowed us to monitor protein enzymatic reactions where nucleotides are modified by alkaline phosphatase and to control the growth of AuNPs using nucleotides as capping ligands.</p> <p>We then investigated the effect of the length of DNA polymers on AuNP surface on AuNP colloidal stability. DNA-modified AuNPs are stabilized electrosterically at a relatively high salt concentration; the removal (or shortening) of the DNA molecules by enzymatic cleavage or the dissociation of DNA aptamers from AuNP surface upon binding to their target destabilizes AuNPs and results in AuNP aggregation. We attribute this to the loss of negatively-charged polymeric DNA molecules that initially served as colloidal stabilizers. This has been applied to the monitoring of enzyme (both protein enzyme and DNA enzyme) cleavage of DNA molecules, and DNA aptamer binding event to its target, respectively.</p> <p> We also studied how DNA polymer conformational changes influence AuNP colloidal stability, which has been employed to monitor DNA aptamer folding events on the AuNP surfaces. We found that AuNPs to which folded aptamer/target complexes are attached are more stable towards salt induced aggregation than those tethered to unfolded aptamers. Experimental results suggested that the folded aptamers were more extended on the surface than the unfolded (but largely collapsed) aptamers in salt solution. The folded aptamers therefore provide higher stabilization effect on AuNPs from both the electrostatic and steric stabilization points of view.</p><p> Finally, we demonstrated the well-defined assembly of AuNPs using long (hundred nanometers to microns) single-stranded (ss) DNA molecules as template in a three-dimensional (3D) fashion. Specifically, these long ssDNA containing repeating units are generated by protein enzymatic reaction (DNA extension through rolling circle amplification) on AuNP surface. The resultant product provides a 3D-like scaffold that can be subsequently used for periodical assembly of complementary DNA-attached nanospecies. </p> <p> We also expect that the facile colorimetric biosensing assays developed in this thesis work provide an attractive means to study biomolecular behaviors (e.g, biorecognition and conformational changes) on the surface, and to investigate other common DNA (or RNA) structural (e.g., triplex, G-quadruplex, hairpin, i-motif) and protein structural transitions.</p> <p> Finally, this thesis work provides some novel and general strategies for the control of nanoassemblies by tuning surface charges and surface-tethered polymers. We expect these principles can also be applied in other AuNP-based sensing platforms that exploit interparticle interactions and in the construction of well-defined nanostructures which involves other types of nano-scaled materials (e.g., quantum dots, nanotubes, nanowires, etc).</p> / Thesis / Doctor of Philosophy (PhD)

Electrolyte interactions with ligand functionalized gold nanoparticles (AuNPs) have broad implication to a wide range of applications in nanoparticle research field. Among a wide range of electrolytes, halides, nitrates, borohydrides, and sulfides are used to study the AuNP interfacial interactions. Although there are many studies on AuNP interactions with anionic species (halides, nitrates, borohydrides, and sulphides), there is limited information on AuNP interactions with metallic cations. Therefore, studying the nanoparticle interfacial interactions with both anionic and metallic cation species is highly important. The research reported here is focused on deepening the understanding of electrolyte interactions with ligand functionalized AuNPs in aqueous solutions. The stability of citrate-residues on AuNPs against ligand displacement has been controversial. In the first study, we demonstrated the direct experimental evidence for the simultaneous adsorption of both citrate-residues and solution impurities onto citrate-reduced AuNPs by using AuNPs synthesized with deuterated citrate in combination with the surface-enhanced Raman spectroscopic (SERS) analysis. The citrate-residues can be readily displaced from AuNPs by a wide range of specific and non-specific ligands including organosulfur and electrolytes. In the second study, we investigated the charge state and the mechanism of silver ion binding onto organothiol functionalized AuNPs. Mechanistic study reveals that silver binding onto AuNPs proceeds predominantly through reactive pathways with proton generations providing the first direct experimental evidence that Ag+ can disrupt the Au-S binding and enhance the mobility of the organothiols on AuNPs. Ligand displacement from AuNPs is important in a wide range of applications. Complete and non-destructive removal of ligands from AuNPs is important and challenging due to the strong Au-S binding and the steric hindrance imposed by ligand overlayer on AuNPs. In the final study, we investigated hydrogen sulphide (HS-), an anionic thiol as an effective ligand to induce complete and non-destructive removal of ligands from aggregated AuNPs. The new insights and methodologies presented in this dissertation are important for studying the electrolyte interfacial interactions with ligand functionalized AuNPs which have a broad impact on nanoparticle surface chemistry.

Doxorubicin is one of the most widely used and effective anticancer agents to treat a wide spectrum of tumors. But its success in cancer therapy is greatly compromised by its cumulative dose-dependent side effects of cardiotoxicity and tumor cell resistance. For the purpose of addressing these side effects, a gold nanoparticles-based anticancer drug delivery system was designed. Five novel thiolated doxorubicin analogs were designed and synthesized and their biological activities have been evaluated. These doxorubicin analogs and the poly(ethylene glycol) (PEG) stabilizing ligands were conjugated to gold nanoparticles via formation of a gold-thiol bond. The systems were evaluated in vitro and in vivo, and the results show that controlled drug release can be achieved either by acidic conditions or by reducing agents in cancer cells, depending on the design of the thiolated drug construct. The overall drug delivery system should achieve enhanced drug accumulation and retention in cancer cells and favorable drug release kinetics, and should demonstrate therapeutic potential and the ability to address some of the current problems of doxorubicin in cancer therapy. / Master of Science

Gold nanoparticles can serve as an ideal radiosensitizer for radiation therapy due to the high-atomic-number nature of gold and the increased tumor specificity in nanoparticle form. The degree of radiosensitization is highly dependent on both the local gold nanoparticle concentration in the tumor and the radiation source type. Previous Monte Carlo simulations have demonstrated that the gamma-ray energy spectrum of Ytterbium-169 is a strong candidate for a high dose rate brachytherapy implementation of gold nanoparticle-aided radiation therapy. Therefore, the current study focuses on the design of a high dose rate Ytterbium-169 source that would maximize dose enhancement during gold nanoparticle-aided radiation therapy; while meeting the practical constraints for the production of a clinically relevant brachytherapy source. Different encapsulation materials are studied in order to determine its effect on the dosimetric characteristics of the source. Specifically, the photon spectra, secondary electron spectra, and dose enhancement characteristics are calculated via Monte Carlo simulations to elucidate the effects on potential radiosensitization during gold nanoparticle-aided radiation therapy. Furthermore, this project involves a study into the modification of external x-ray beams from a Philips RT-250 orthovoltage x-ray machine in an attempt to match the dosimetric characteristics of the Ytterbium-169 brachytherapy source. This investigation will enable the production of an external beam that can serve as a good surrogate of an actual brachytherapy source and facilitate the pre-clinical investigation of gold nanoparticle-aided radiation therapy with Ytterbium-169.

Vaccination is one of the most effective public health interventions in the world, saving millions of lives and preventing the onset of debilitating diseases. With widespread emergence of multi-drug resistant pathogens, the importance of preventative medicine has become even more apparent. However, one of the limiting factors in developing novel vaccines that are both safe and highly immunogenic is the availability of adjuvant delivery systems licensed for human use. The purpose of this study was to investigate the role gold nanoparticles could play as an effective vaccine delivery system. A variety of coupling chemistries were explored for their ability to conjugate protein and polysaccharide antigens onto the surface of gold nanoparticles for the development of vaccines against a number of biologically important human pathogens including Y. pestis, B. mallei and S. pneumoniae. Retention of antigenicity and coupling efficiency of conjugated molecules was measured using characterisation techniques such as localised surface plasmon resonance and immunoblotting. Gold nanoparticle coupled antigens were then used to immunise mice and to measure the protective efficacy and the immunological response induced. The findings indicate antigen-specific immune responses are elevated when an antigen is coupled onto gold nanoparticles. Moreover, immunological data from nanoparticle coupled glycoconjugate vaccines against B. mallei and S. pneumoniae indicate the likely presence of a strong T cell immune response which is essential for providing immunological memory. Finally, an intracellular trafficking assay was carried out to identify some of the mechanisms that might be involved in uptake of gold nanoparticles into professional phagocytes. Confocal imaging of receptors associated with endosomal compartments revealed that gold nanoparticles may enter cells through multiple pathways. The findings reported in this study suggest that gold nanoparticles may be an excellent candidate for further investigation as a novel vaccine delivery system.

Cancer is one of the most notorious diseases affecting the human population today with very few effective treatments. Due to the disparate nature of cancers, it is difficult to obtain a treatment that can cure cancer. Thus, there is a large influx of research towards cancer therapies, leading to one of the discovery that cancer cells (tumors) have a low thermotolerance in comparison to normal cells. If the temperature of the cancer cells is increased into the hyperthermia range (~45°C) thermal damage occurs, causing cell death by protein denaturation and membrane disruption. A recent development in this field has been in the photothermal treatment of tumors, which is starting to utilize plasmonic particles to enhance the specificity of the treatment. The plasmonic nanoparticles, specifically gold, can reach the tumor site using passive targeting and when irradiated with a tuned laser will emit heat localized to a small region around the nanoparticle killing the surrounding cancer cells. This process has been shown to reduce tumor size in vivo with gold nanoshells and gold nanorods. However, it has not been shown which particle is better at delivering the heat to the tumor site. Therefore in this study, it will be shown which particle generates the most heat. Solutions of tissue simulating phantom and different concentrations of nanoparticles were irradiated with a laser to measure the increase in temperature. Additionally, simulations were performed using Mie Theory for nanoshells and the Discrete Dipole Approximation for nanorods. Based on the physical parameters of the nanoshells and nanorods used in this experiment, the adjusted absorption cross-section was determined. It was found that nanoshells generated the most amount of heat on a per particle basis, and that it was necessary to have a nanorod concentration of 5.5 times the concentration of nanoshells to generate the same amount of heat as nanoshells. These results were confirmed using Monte Carlo and Finite Difference Modeling of the nanoparticle heating experiments. However, the choice of nanoparticle still depends on the application and the targeting efficiency in vivo. / text

Glycerol is a very promising alternative fuel to hydrogen in fuel cells. However, the utilisation of glycerol as a fuel requires a good catalyst, due to the slow kinetics of glycerol electrooxidation. Gold has been identified as a promising catalyst due to its high activity and stability for glycerol electrooxidation – although the overpotentials are higher than on platinum and palladium. Modification of a nano-Au/C catalyst by the addition of MnO2, in an attempt to further improve the activity and lower the overpotential for glycerol oxidation, was therefore first explored. This was followed by investigations into the effects of gold particle size and loading. Finally, the effect of gold particle size on oxidation of gold-catalysed glycerol oxidation intermediates was also briefly explored. Studies into MnO2 addition showed that the pre-deposition of MnO2 yielded catalysts with smaller, more uniform gold particles, and catalysts with MnO2 contents of 5 and 9 wt % had higher mass activities and lower onset- and peak- potentials than Au/C. All the Au/xMnO2/C catalysts were more active than the palladium- and platinum-based catalysts reported in literature, which effectively demonstrated the advantage of using a gold-based catalyst for glycerol oxidation – especially when supported by MnO2 which lowered the overpotential for glycerol oxidation over gold. For the study into gold particle size, small gold particles of average diameter ≤ 4.7 nm had higher gold mass-based activities than medium-sized (14.7 nm) particles and were at least twice as active as catalysts containing large (≥ 43 nm) gold particles. The small gold particles also gave lower glycerol oxidation onset potentials, which was attributed to the predominance of Au(110) planes on those particles. Glycerol oxidation also appeared to proceed further along the oxidation pathway over small gold particles, which was confirmed in preliminary studies into the oxidation of glycerol oxidation intermediates. However, specific activity increased with increasing gold particle size, due mainly to the higher intrinsic activity of the Au(111) plane, which increased relative to Au(110) with increasing gold particle size. The important requirements for fuel cell applications are factors such as high mass activity, low overpotentials and high stability – all of which were met by the catalysts containing small gold particles defined by predominantly Au(110) facets. Investigations into the gold loading effect showed similar mass- and specific- activities for catalysts with 5-20 % gold loading. However, only the catalysts with higher gold loadings (15-20 %) did not deactivate early during CV, indicating that a larger gold surface area is necessary to resist poisoning at high potentials. On the basis of low onset potentials, high mass activity, and stability at low overpotentials, a minimum gold loading of 12.5 % appears to be necessary for a supported gold catalyst with small gold nanoparticles; although even higher loadings may be preferable for a higher power output in a fuel cell. Importantly, the insights gleaned from this study on the fundamental properties required for early activation, activity and stability of the gold catalysts could lead to a more intelligent design of gold-based catalysts in future.

The optical properties of gold in the visible are dominated by the response of the free conduction electrons to light. In gold nanostructures, the surface charge density adopts a configuration that is constrained by the shape of the nanoparticles. As a result, the scattering of light by gold nanoparticles exhibits a resonant response characterised by a strong scattering and absorption in a narrow range of frequencies. The spectral range of this \emph{localised surface plasmon resonance} (LSPR) can be tuned by varying the size and shape of the gold nanoparticle --- the nanoparticles act as nanoscale antennas for the visible light. Confirmation of this scaling rule is obtained by conducting experiments with nanoparticles of varying size and aspect ratio. Such particles are fabricated by electron-beam lithography, and characterised by dark-field spectroscopy. Not only does the LSPR shift in frequency with a change of particle size, but its spectral lineshape is also modified. The intensity and width of the LSPR are dictated by a variety of factors that are related to the intrinsic material properties (the complex dielectric function of gold), and to the particle geometry and environment. The optical response of small gold nanorods is well described by a simple oscillating dipole model --- the incident electromagnetic field induces a current in the particle that re-radiates light (scattering). A series of refinements can be made to model more accurately the optical response of realistic particles. If the dipole moment characterising the particle is allowed to vary in phase across the particle, retardation effects provide a correction for the effective dipole moment of the particle. As the particle size approaches the wave length in the surrounding medium, the dipolar approximation breaks down and higher order multipoles need to be considered. The Mie theory provides a very accurate description of the response of spheres of arbitrary size. Further, the T-matrix and other numerical techniques can be employed to accurately reproduce the scattering properties of particles of arbitrary shapes. When the scattering sample consists of a collection of gold nanoparticles, the collective optical response is affected by two key factors. First, the measured LSPR is a convolution of the distribution of particle sizes with the individual response of a single particle. This leads to an inhomogeneous broadening of the LSPR lineshape. Second, the light that is scattered by one such particle near resonance can strongly affect its neighbours which scatter light in proportion to the net field they experience, that is the sum of the incident field plus the perturbation arising from the neighbouring particles. The onset of such multiple scattering events is observed even for particle separations that are several times larger than the particle size. Several regimes of interaction can be distinguished according to the ratio separation / wavelength. First, when the particles are in close proximity (separation $\ll$ wavelength), near-field interactions dominate and result in a spectral shift of the LSPR accompanied with a spectral broadening. Second, when the separation is commensurate with the wavelength, a coherent interaction can develop that couples a large number of particles. In ordered arrays, such coupling gives rise to a geometrical resonance that can strongly affect the LSPR of the particles. In particular a sharp spectral feature is observed that depends on both the single particle response and the geometrical arrangement of the particles in the array. The coherence of such multiple scattering in diffractive arrays of gold nanoparticles can be broken by introducing disorder in the distribution of particle sizes, or in the particle positions. The optical properties of an irregular array reflect the departure from a periodic system and the spectral lineshape evolves as the level of disorder is increased. In the limit of uncorrelated positions, the diffractive coupling is suppressed and the response of the collection of the particles rejoins the response of isolated particles.

The handling of metallic nanoparticles often requires their dispersion into several polar and nonpolar solvents. Solid-phase stages or polymer-based ligands are commonly required to complete the transfer. The construction of a thiol ligand based in oleic acid, and its ability to efficiently assist in gold and silver nanoparticle aqueous-organic phase transfer is reported. After the transfer, the particles are completely dispersed in an organic solvent, preserving their diameter and morphology, as confirmed by ultraviolet-visible spectroscopy and scanning transmission electron micrographs.