Mechanism of toxicity of aluminium-based adjuvant (ABA) nanomaterials

There is a burgeoning interest in human exposure to what have been called nanoparticulates. It has become
apparent that sub-micron sized particles are able to enter the body and potentially accumulate in tissues and
organs. These nanomaterials are both novel materials which are being developed for myriad purposes and
they are materials which are 'naturally' present in many different environments as the products of both
natural and man-made phenomena. While there is evidence that such nanomaterials are becoming more
widespread it is also clear that we understand very little about their potential modes of toxicity. Actually one
such form of nanomaterial has been used by humans for decades and are the aluminium-based adjuvants
which are used in approximately 80% of all human vaccinations. While these materials are highly biologically
reactive we still do not understand fully how they work and we believe that they would be an extremely
useful model system for understanding the toxicity of nanomaterials. The objectives of our research are to
obtain a thorough understanding of the physical and chemical properties of these nanomaterials and then
investigate how these influence their biological reactivity. We hope to explain how they work as adjuvants
and in doing so we will help in the design of future safe and effective adjuvant materials. In addition we are
hoping to optimise the toxicity of such materials such that they might be used to kill tumour cells in cases of
malignant brain cancer. None of these objectives can be achieved without an understanding of the
mechanisms of toxic action of these nanomaterials and this is the primary objective of this research.

Aluminium-based adjuvants (ABA) are micron-sized materials which upon dissolution in interstitial fluid form nanometer-sized particles with biological effect. Their mechanism of action in improving the immune response remains to be fully elucidated and these nanomaterials are excellent models for understanding nanotoxicity. The latter is important in that the number and amount of nanomaterials to which humans are exposed during their everyday lives are burgeoning without any sure knowledge of their ultimate biological fate and safety. Understanding of the mechanisms of toxicity of nanomaterials in humans could be determined from a thorough understanding of how ABA enhance the immune response. In preliminary research in which phagocytosing cells were exposed to ABA we identified mitochondria as potential targets of ABA
mediated nanotoxicity. Cell fractionation suggested that mitochondria accumulate ABA nanomaterials. Herein we combine a thorough examination of the physico-chemical properties of both current and novel ABA with detailed investigations of their biological effect in phagocytosing cells and in an established rodent model of malignant brain tumours. Physico-chemical characterisations will enable an understanding of the bioinorganic chemistry of these materials in biological milieu which will be used to understand their cellular toxicity and ultimately their application to adjuvant-based immunotherapy. We will apply state-of-the-art fluorescent labelling methods such that we will be able to follow the trafficking of ABA nanomaterials both in cell culture and in an animal model. Once a detailed understanding of the toxic mode of action of ABA is obtained the idea is to optimise similar materials and exposure regimes both for the effective and safe use of ABA in vaccinations programmes but also for targetting cell death in malignant brain tumours which could become a therapeutic off-shoot of an improved understanding of the toxicity of ABA nanomaterials.

While the research described in this proposal is fundamental it is also applied in that it will address current and novel adjuvants used in vaccination programmes. Through a thorough understanding of the mechanism by which nanomaterials in ABA stimulate an immune response we will provide essential information which could be applied to the next generation of ABA making them both more effective and, importantly, safe to
use. Equally important we will also greatly improve our understanding of how nanomaterials exert toxicity in vivo. The latter, will also help us to determine the potential value of adjuvant-based immunotherapy for brain tumours. Can we design ABA nanomaterials such that their toxicity can be focussed on killing tumour cells? The immediate beneficiaries of our research are likely those involved in the design of new adjuvant materials
for vaccination programmes and immunotherapy. ABA are the most cost effective form of adjuvant and are particularly important where antigen is scarce such as in mass vaccination programmes. While ABA are widely used today their use is not without some controversy and such probably comes from our lack of understanding as to their modes of action. We will add considerably to this knowledge base and in doing so
demonstrate how nanomaterials can actually be applied positively and safely. Other indirect beneficiaries will be those charged with making policy relevant to human exposure to nanomaterials in the everyday and workplace environment. The burgeoning use of novel nanomaterials as well as exposure to such from other sources such as air pollution and even volcano plumes (!) makes an understanding of the mechanisms of
toxicity of such absolutely critical to avoid potential future problems of uncontrolled exposure. Once again, we will provide strong indicative data outlining possible mechanisms of the toxicity of such materials and this should benefit both those involved in adding to human exposure and those who have to regulate against such exposure. This research proposal will contribute towards MRC s policy of high quality training for research staff in that the PDRA will receive a wide and diverse range of training in the 3 different research environments which
constitute this project.