The Interaction of Synthetic Nanoparticles with Biological Systems

Gysell Mortimer (2011). The Interaction of Synthetic Nanoparticles with Biological Systems PhD Thesis, School of Biomedical Sciences, The University of Queensland.

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Author Gysell Mortimer
Thesis Title The Interaction of Synthetic Nanoparticles with Biological Systems
School, Centre or Institute School of Biomedical Sciences
Institution The University of Queensland
Publication date 2011-01
Thesis type PhD Thesis
Supervisor Prof. Rodney Minchin
A/Prof. Darren Martin
Total pages 173
Total colour pages 29
Total black and white pages 144
Subjects 11 Medical and Health Sciences
Abstract/Summary Abstract Many nanomaterials are currently under development for use in biomedical applications including drug delivery, tissue imaging and as property enhancers in medical devices. Nanoclays, such as synthetic hectorite (HECT) and layered double hydroxides (LDH) are already widely used in various industrial applications and consumer products. Furthermore, these nanoclay materials are currently under development for use in medical composites and devices. However, our understanding of how nanoparticles interact with cells and tissues is limited. This has raised safety concerns about the consequences of human exposure to nanoparticles. Several recent studies have shown that the disposition of various nanoparticles in biological systems can be influenced by particle size, shape, and surface characteristics. However, very little is known about how HECT and LDH nanoparticles interact with human cells and whether they are likely to generate any adverse effects. Initially, we used several cell-based assays to study the cellular response to HECT and LDH exposure and found that the presence of plasma proteins can cause marked changes in nanoclay-induced cytotoxicity. Nanoclay exposure in the presence of plasma proteins did not alter mammalian cell viability, the plasmatic phases of coagulation or stimulation of cytokines from peripheral blood mononuclear cells. However, toxicity towards erythrocytes was evident in the absence of plasma proteins. When erythrocyte exposure to the nanoclays was studied in the presence of proteins, a clear protective mechanism against toxicity was observed. In addition, the extent of protection varied with the type of protein used. These results showed that interactions with proteins may be critical in determining biological responses towards nanoclays. The cellular internalisation of nanomaterials is of particular interest to the nanoscience field due to the potential to interact further with internal components of the cell. We developed methods to fluorescently label both nanoclays that enabled the study of particle interactions within cells. Although fluorescent labelling for LDH has been previously published, our method achieved greater fluorescence intensity and improved stability. The fluorescent labelling of HECT accomplished in this study is a novel method allowing the use of a family of dyes with varying emission wavelengths. The ability to fluorescently label these nanoparticles allowed us to investigate whether they were able to enter cells. LDH was internalised by HeLa cells, which is consistent with several other studies reporting the ability of LDH to enter cell types by clathrin-mediated endocytosis. By contrast, HECT was internalised only by macrophage-like cells (differentiated THP-1 cells, dTHP-1). To investigate this further, the study was directed to establish the transport mechanism of HECT. We showed that HECT uptake in dTHP-1 cells occurs by an endocytic mechanism that was affected by the presence of serum. Because HECT uptake only occurred in macrophage-like dTHP-1 cells, but not monocytic THP-1 cells, phagocytic internalisation was considered a likely mechanism. By using a number of inhibitors selective for particular phagocytic receptors, we determined that scavenger receptors were involved in HECT uptake. However, the use of these inhibitors in medium without serum suggested that the mechanism could change from scavenger receptor-mediated in the presence of serum, to mannose receptor-meditated in the absence of serum. The adsorption of serum proteins to nanomaterials has recently been emphasised as the surface that tissues and cells recognise. By using two-dimensional gel electrophoresis, we found that HECT bound numerous proteins of which albumin appeared the most abundant. When serum in the culture medium was replaced with albumin, uptake was inhibited by scavenger receptor inhibitors. These results indicated that adsorbed albumin may be important in facilitating HECT uptake by scavenger receptors. Since recognition of albumin by scavenger receptors only occurs when albumin is modified, we propose a model were albumin may undergo structural alterations induced by binding to HECT. By monitoring the shape of albumin with far UV circular dichroism, we have shown that the addition of HECT to albumin produces a loss in protein secondary structure or unfolding of the protein. This change in protein conformation is distinct from other well known chemical modifications to albumin that bind scavenger receptors. Therefore, we propose that this unfolding of albumin reveals structure-specific epitopes recognised by scavenger receptors. Various subclasses of the scavenger receptor family recognise different modified proteins. Our investigations using scavenger receptor AI, BI and MARCO, determined that uptake of albumin-adsorbed HECT is class A specific. Collectively, the research presented in this study shows that HECT and LDH produce biological responses that are protein dependent. Protein specific configurations produced by adsorption may provide predictive biodistribution information in vivo.
Keyword Nanotechnology
Cell Uptake
Scavenger Receptor
Protein adsorption
Additional Notes Colour Pages of pdf file: p20 -(labelled as p13 in thesis) p21 -(labelled as p14 in thesis) p23 -(labelled as p16 in thesis) p24 -(labelled as p17 in thesis) p26 -(labelled as p19 in thesis) p31 -(labelled as p24 in thesis) p33 -(labelled as p26 in thesis) p39 -(labelled as p32 in thesis) p41 -(labelled as p34 in thesis) p76 -(labelled as p69 in thesis) p88 -(labelled as p81 in thesis) p90 -(labelled as p83 in thesis) p91 -(labelled as p84 in thesis) p92 -(labelled as p85 in thesis) p94 -(labelled as p87 in thesis) p95 -(labelled as p88 in thesis) p100 -(labelled as p93 in thesis) p102 -(labelled as p95 in thesis) p103 -(labelled as p96 in thesis) p112 -(labelled as p105 in thesis) p114 -(labelled as p107 in thesis) p115 -(labelled as p108 in thesis) p116 -(labelled as p109 in thesis) p123 -(labelled as p116 in thesis) p126 -(labelled as p119 in thesis) p130 -(labelled as p123 in thesis) p139 -(labelled as p132 in thesis) p167 -(labelled as p160 in thesis) p171 -(labelled as p164 in thesis) Landscape pages: p130 -(labelled as p123 in thesis) p139 -(labelled as p132 in thesis)

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