Nanoparticle Cytometrics: a quantitative analysis of the toxic effect of nanoparticles

Lead Research Organisation: University of Leeds
Department Name: Institute of Materials Research

Abstract

The last decade has seen an explosion in the development and use of nanoscale technologies and materials and this trend will undoubtedly continue for some time to come. It is clear therefore that human exposure to nanoscale particles will grow dramatically and this has triggered serious concerns over the safety of nanoparticles in relation to human health and the wider ecosystems within which we live. Much of this stems from the paradigm shift incorporated in the move to nano scales which invalidates current chemical risk assessments that are based on bulk properties and hence grossly inappropriate for nano-systems. A marked example of this is provided by noble metals such as Gold; these are traditionally viewed as inert and hence bio-compatible however in nano-particulate form the huge increase in their surface area makes them highly reactive. The issue of surface area has led to the realisation that the quantisation of potential toxins into nano-particulate form is important and that assessment of health risk must take account of specific particle number rather than gross measures of chemical solution based on weight per volume. There is also a growing realisation that the function of particles due to size, shape and chemistry is important. This gap in understanding of chemical hazard at the nanoscale has led to a number of reports from government agencies and royal commissions emphasising the need to develop new measurement techniques to quantify the level of nanoparticle dose and to assess potential toxicity. We propose to use flow cytometry, a laser-based technology, to optically track fluorescent nanoparticles within populations of living cells. This optical measurement approach will be linked to electron microscopy to image particles within cells at the nanoscale. Together these techniques will provide a fully calibrated metrology providing information on number of particles per cell for each and every cell within a measured set (typically a million cells). Our objective is to provide a fundamental understanding of the way in which nanoparticle dose is acquired by cells through natural uptake mechanisms (endocytosis) and then diluted within growing tissue as particles are divided between daughter cells upon cell division (mitosis). A range of cell types will be used to reflect the main exposure routes to nanoparticles, i.e. skin, lung and circulatory (blood) systems. A detailed study of any toxic effects of nanoparticles on the cells will be undertaken and this together with the absolute quantification of dose will allow us to correlate any potential nanotoxicity to the number and form of nanoparticle within a given cell type.

Publications

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Brown A (2013) Nanomedicine

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Brydson R (2015) Microscopy of nanoparticulate dispersions. in Journal of microscopy

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Hondow N (2014) The use of transmission electron microscopy in the quantification of nanoparticle dose in Journal of Physics: Conference Series

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Manshian BB (2015) Cell type-dependent changes in CdSe/ZnS quantum dot uptake and toxic endpoints. in Toxicological sciences : an official journal of the Society of Toxicology

 
Description Key findings
1) We have used an electron microscope to measure the dispersion of nano particles in liquids (by rapidly freezing the liquid with the nano particles trapped in the suspension). This has enabled measurement of nano particle clustering in challenging liquids such as cell culture media which contain other suspended solids. Using the method we can establish whether individual or groups of nano particles are delivered to cells for toxicity texting or cell uptake studies.

2) We have used electron microscopy to count the number of fluorescent nano particles taken up by cells. By combining this count with the fluorescence intensity measured from many cells in a population we can estimate the average number and range of nano particles taken up by each cell across a cell population. This measure of dose will help us to predict the potential efficacy of a nano particle based medicine.

3) We have used electron microscopy to confirm that nano particles which fluoresce with different colours can be taken up separately by cells. This is a key step in a new method we have developed to accurately mark and track live cells over several hours.

4) We have assessed the toxic potential of semiconductor particles that fluoresce (and can be used as optical biomarkers) as a function of the surface charge on the particles. We suggest that negatively charge particles produce the most efficient uptake with lowest toxicity.
Exploitation Route Others will use the microscopy techniques developed in this work for similar nano toxicology and nano medicine investigations.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description So far our findings have been communicated by publication, at academic conferences (including for invited talks) and by presentation to regulatory bodies (the EU grant meetings) and external companies, such as AstraZeneca. We would expect other researchers to use the microscopy techniques developed in this work for similar nano toxicology and nano medicine investigations. The post-doctoral researcher employed on the contract has been awarded an independent research fellowship (AXA award and now a University Academic Fellowship).
First Year Of Impact 2011
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal