Design and synthesis of nanomaterials as combination therapeutics

Abstract

Integrating nanomaterials with biological entities has led to the development of biotechnology-derived therapeutic-enhancing products. However, to optimise the design of hybrid bio-nanomaterials, it is essential to understand their physicochemical properties and interactions on the molecular level. The primary aim of this thesis was to understand how the design of hybrid-nanomaterials affects their interaction and internalisation by cancer cells, and ultimately their behaviour on tumour accumulation in vivo. By controlling the antibody density of mixed-micelles, the aim was to address key questions relating to optimisation of their function related to the effect of ligand density. This approach gives fundamental insight into how these parameters affect the rate and mechanistic pathways into cells as well as a method to probe the intricate interplay between increased targeting efficiency versus the subsequent immune response.Chapter 1 discussed an overview of Cancer nanomedicine along with the current opportunities and challenges. This includes different factors affecting the efficacy of nanomedicines. Chapter 2 focused on an in-depth review of the versatility of conjugation approaches involved in the synthesis of antibody-nanoparticle conjugates. While such conjugates show significant promise as next-generation targeted nanomedicines, it is recognised that there are no clinically approved targeted-nanotherapeutics on the market. This fact is reflected upon within this chapter, and attempts are made to draw some reasoning from the complexities associated with the bioconjugation chemistry approaches that are typically utilised.Chapter 3 discussed the synthetic methodology and characterisations used for the development of polymer-antibody conjugates. Firstly, for synthesising the polymer-antibody conjugate, the coupling efficiencies of two different click chemistry approaches, Copper-catalysed Azide-Alkyne Cycloaddition (CuAAC) and Strain Promoted Azide-Alkyne Cycloaddition (SPAAC) have been compared, as well as the effect of reactive group positioning on the linear polymer chain (R- and Z- group modification with dibenzocyclooctyne (DBCO). CuAAC coupling of the azide-functional antibody onto the polymer demonstrated lower efficiency compared to SPAAC. However, the presence of the DBCO functional group alone does not necessarily ensure efficient antibody conjugation; its position on the linear polymer chain has an important effect. Here, DBCO incorporation using R-group modification successfully facilitated antibody conjugation, but this was limited by the polymer chain with broad molecular weight distribution due to poor control over polymerisation by the chain transfer agent (CTA).Chapter 4 further built upon the results and observations in Chapter 3. In this chapter, an acid functionalised CTA was used to synthesise the polymer, which resulted in the formation of controlled polymers with narrow molecular weight distribution. Following polymerisation, the acid group was modified with DBCO amine, resulting in the formation of polymers with controlled DBCO functionality in the R-group. These polymers showed better conjugation with antibodies and Polyethylene glycol (PEG), which was validated by High-performance liquid chromatography (HPLC) and cellular studies. Both the antibody- and PEG-polymer conjugates were then mixed in different ratios to make mixed-micelles with varying ligand densities. Chapter 4 further discussed the size and the stability of mixed-micelles under varying factors including temperature and solvent conditions.Chapter 5 discussed the cellular response of mixed-micelles, mainly focusing on the influence of ligand density on cellular association and uptake. This was analysed by flow cytometry analysis, and the intracellular distribution of these micelles was validated by confocal microscopy. The performance of mixed-micelles in vivo was also analysed using optical fluorescence imaging. It was hypothesised that the increase in ligand density on mixed-micelles leads to an increase in tumour uptake to a maximum, followed by a decrease due to triggered immune response, which was proven in the current study. Specifically, information on the distribution of micelles into prostate cancer cell lines within a mouse model was derived from fluorescence imaging of both the whole animal as well as the individual organs, particularly the liver and spleen. In addition, the distribution of micelles within the murine liver and spleen immune cells was analysed using flow cytometry with different immune cell markers. Finally, to demonstrate the translatability of these materials into human subjects, the response of the polymeric micelles with varying antibody densities was studied against human white blood cells, one of the primary components that interact with exogenous materials when injected intravenously into humans.In summary, the work presented in this thesis demonstrates a facile methodology to precisely control the ligand density within micelles through self-assembly, and they were used to compare the efficiency of cellular and whole animal targeting of human prostate cancer. This is an important consideration that is often not considered until late in a study; here, all in vitro findings were validated with in vivo data to correctly inform on the properties that affect nanoparticle targeting in the whole animal. By understanding the basic science of nanomaterial interaction with biological entities, the ability to apply this technology to more demanding systems is enabled for the wider therapeutic delivery and diagnostic fields.