Tailored synthesis of ultra-small nanoparticles for biomedical applications

Accurate and early diagnosis of disease is a critical worldwide challenge in healthcare. The emergence of nanomaterials has opened up the possibility of engineering new and improved diagnostic and therapeutic tools which can interact on a sub-cellular level. Ultra-small (< 3 nm) metal-oxide nanoparticles (NPs) have been highlighted as particularly strong candidates for enhancing positive T1 image contrast in magnetic resonance imaging (MRI), an important and powerful diagnostic imaging tool. Current T1 gadolinium contrast agents are limited by long-term toxicity issues, and cannot be given to patients with kidney disease, which represents 16% of the UK population. Metal-oxide NPs, such as iron oxide, are more biocompatible, and below 3 nm, they present the ideal magnetic properties to enhance T1 image contrast, offering a potential alternative to current gadolinium-based agents.
However, several roadblocks must be overcome to reach clinical application of ultra-small NPs. The primary challenge lies in the development of suitable synthesis methods that offer the exquisite control over particle size and composition that is required to produce ultra-small NPs. Furthermore, their properties need fine-tuning in order to achieve T1-weighted contrast that is more comparable with gadolinium. An additional important consideration is that the NPs are designed with core and surface features that enable excellent stability, performance and biocompatibility in vivo.
This thesis details the synthesis, characterisation, and functionalisation of ultra-small iron oxide and cobalt ferrite NPs for application as T1 MRI contrast agents. The main findings of this thesis concern the investigation of 2.4 nm cobalt ferrite NPs for T1 MRI. Cobalt ferrite NPs may be excellent candidates for T1-weighted contrast as they offer beneficial properties in comparison to iron oxide, such as lower magnetisation and greater chemical stability.
Size-controlled synthesis of ultra-small NPs was addressed with a simple and fast thermal decomposition approach using oleylamine and acetylacetonate precursors. Sub-nm size control in the range of 2.4-5 nm was achieved by tailoring the oleylamine-to-precursor ratio. Annular dark-field scanning transmission electron microscopy revealed highly-crystalline cubic spinel particles 2.4 nm in size with atomic-resolution. Both iron oxide and cobalt ferrite NPs displayed size and coating-dependent superparamagnetic properties.
In consideration for in vivo applications, two strategies were explored to obtain water-dispersible NPs. A novel phase transfer method with bovine serum albumin proved unsuitable as the NPs quickly flocculated. Ligand exchange with dimercaptosuccinic acid rendered the particles stable in physiological conditions with an average hydrodynamic diameter of ~12 nm. It was found that dialysing the particles prior to ligand exchange to remove excess oleylamine and alkalising the particles after ligand exchange were key steps to obtain reproducible results.
The potential toxicity of the NPs was investigated with cytotoxicity and oxidative stress assays comparing the effect of particle size and metal composition. Neither iron oxide nor cobalt ferrite NPs of any size displayed cytotoxicity towards HepG2 liver cells. However, both NP types produced reactive oxygen species (ROS), and it was found that cobalt ferrite produced significantly more. Serendipitously, ROS generation was effectively alleviated through conjugation to a bile acid derivative, cholic acid. Conjugation was achieved by EDC-NHS coupling, following which the ROS levels reduced by 62% and 67% for iron oxide and cobalt ferrite NPs respectively. This new insight reveals the potential of cholic acid to mitigate ROS generation, a common issue encountered in metal-oxide NPs. Furthermore, the cholic acid coating could promote active liver-targeting in vivo.
Finally, the MRI application of DMSA-coated iron oxide and cobalt ferrite NPs was assessed with phantom studies using 3.0 T and 9.4 T scanners. Cobalt ferrite NPs were found to have a low r2/r1 ratio suitable for a T1 contrast agent. The work described in this thesis demonstrates the potential of ultra-small ferrite NPs towards application as liver-targeted T1 MRI contrast agents. The synthesis and functionalisation methods described may open up the possibility of applying ultra-small ferrite NPs in other exciting areas of biomedicine.

Keywords: MRI
Nanoparticles
Contrast agents
Nanomedicine
Advisor: Grobert, N