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Vol. 34 No. 4
July-August 2012

Size Matters: Measurement Helps Solve Nanoparticle Toxicity Challenges

Lord Kelvin was famously quoted as saying ". . . when you can measure what you are speaking about, and express it in numbers, you know something about it; but when you cannot express it in numbers, your knowledge is of a meagre and unsatisfactory kind . . . ."

Nanoparticle Tracking Analysis (NTA) in combination with FFF-ICP-MS characterized particles for size and elemental composition. Image courtesy of Andrew Brookes, www.andrewbrookes.co.uk.

In case you missed it, World Metrology Day1 was observed on 20 May 2012, celebrating the signing of the Metre Convention by representatives of 17 nations 137 years ago. The Metre Convention created the International Bureau of Weights and Measures and set the framework for global collaboration in the science of measurement and its application in industry, innovation, and society. The worldwide uniformity of measurement remains as important today as it was in 1875.

World Metrology Day has become an established annual event during which more than 80 states celebrate the impact of measurement on our daily lives—an important, though not always immediately apparent, aspect of modern society. This year, the theme was "Metrology for Safety," a topic that concerns us all in a variety of commonplace ways. One such concern has emerged from the field of nanomaterials: Measurement procedures must be developed for establishing the potential toxicity of nanoparticles in consumer products and their effect on humans and the environment.

Nanoparticles are already incorporated in over 1300 commercial products,2 from food and consumer products to electronics, automotive parts, and medical products. With the 2012 Olympic Games fast approaching, news of sports clothing containing nanoparticles has hit the headlines. Such materials offer the ability to produce lighter, stronger, or more streamlined athletic clothing (e.g., swimsuits with low levels of drag, running spikes with carbon nanotube reinforced plates, and sunglasses with a protective and anti-reflective polymer "nanofilm"). Our athletes are now kitted out in sportswear that not only looks great but also performs in a different league.

. . . the unique properties of nanoparticles make measuring their potential toxic effects more problematic.

While the applications for nanomaterials in clothes are far ranging, the wider field of nanotechnology offers the potential to provide solutions to our biggest global challenges. For example, nanotechnology may provide a sustainable energy solution in terms of energy conversion, storage, and conservation. Nanotechnology may also offer better, more cost-effective medical treatment through improved drug delivery.

The increased use of nanotechnologies is driven by the unique mechanical, thermal, and catalytic properties that materials develop when structured at the nanoscale. However, it is these unique properties, while beneficial for technological innovation, which could also make nanomaterials toxic to biological tissues, raising concerns that they pose a risk to the environment and human health. The potential benefits of nanotechnology can only be fully realized if nanomaterials, particularly those for use in nanomedicine applications and consumer products, are shown to be non-toxic. Limitations in our knowledge of potential toxicity are partly due to the lack of methodology for the detection and characterization of nanoparticles in complex matrices. Therefore, in order to address this issue, the development of robust methods to provide adequate toxicity data, comparable between laboratories, is required.

The Challenge

As the name suggests, nanoparticles are very small—less than 100 nm, which means they could overcome some of the body's defense mechanisms, such as the cellular barriers, which protect against foreign objects. In order to take full advantage of the technological benefits of nanotechnology and to sustain competitive economic growth, it is becoming increasingly necessary to ensure that nanoproducts are safe at all stages of their life-cycle and that the public and the environment is adequately protected from any adverse effects.

Current methods for understanding the potential harmful or toxic effects of nanoparticles rely on the well-developed in vitro and in vivo testing processes developed for assessing chemical, pharmaceutical, and consumer products. While comparisons can be drawn from these testing regimes, the unique properties of nanoparticles make measuring their potential toxic effects more problematic. As a consequence, it is difficult to predict the effects of nanomaterials on human health based on known risks for macrosized particles with the same chemical composition.

In addition, measuring the potential toxicity of nanoparticle-containing consumer products is often complicated by the complex matrix of the food or product. There is currently a lack of methodology for the reliable characterization of inorganic nanoparticles added to food, and a lack of knowledge about the stability of such materials. Therefore, analytical methods are needed that will enable accurate element quantitation and rapid size characterization of nanoparticles in consumer products.

The principle of FFF (field-flow fractionation).
Image courtesy of Postnova Analytics, www.postnova.com

Method Development for Nanoparticle Extraction

Titanium dioxide is used predominantly as a white pigment in a variety of products, including coffee creamers, toothpastes, and sunscreens. In sunscreens, its high refractive index protects the skin from UV radiation from sunlight. Initially, titanium dioxide was considered to be an inert mineral, non-toxic to humans and the environment. However, following its broad application, concerns have arisen that its toxicological risk has not been investigated sufficiently.

The accurate detection and identification of nanoparticles in food and consumer products requires an efficient sample preparation and extraction process to separate nanoparticles from the complex matrix of the product. Of the family of separation methods, field flow fractionation (FFF) is one of the most used and appropriate techniques for separation of nano-objects in complex matrices. FFF provides separation prior to detection and characterization of nanoparticles. The separation process is similar to chromatography except there is no stationary phase and separation is based on physical forces as opposed to chemical interactions. Separation occurs in a thin flow channel where the particles are forced downwards by the cross flow. Smaller particles will diffuse back into the channel flow more quickly, and will elute faster than larger particles (see figure above). By developing methods involving the coupling of FFF to inductively coupled plasma mass spectrometry (ICP-MS) and multi-angle light scattering (MALS), the technique has the potential to provide size-resolved data on the elemental composition of nanoparticles, which is critical to environmental and toxicological investigations of nanomaterials.

Using these techniques, LGC, the UK's designated National Measurement Institute for chemical and bioanalytical measurement, has developed a new method for the characterization of titanium dioxide particles in sunscreens which involves the development of an improved extraction method for nanoparticle isolation.3 This research forms the first systematic comparison and optimization of extraction methods for titanium dioxide nanoparticles in sunscreen samples. Sunscreens were selected for this research due to their wide use, high fat content, and highly complex matrix.

Previous published research has demonstrated the applicability of coupling FFF to ICP-MS in order to characterize nanoparticles.45 LGC is building upon this research by using a labelled titanium dioxide reference material as a spiking material for quality control and comparability. The novel approach of sample spiking with aluminium-labelled reference particles of known size enables the effect of extraction and separation conditions on particle size distribution to be studied. The sunscreens were analyzed and compared for titanium extraction efficiency, particle size distribution, and titanium dioxide recovery from the FFF channel. Using FFF-ICP-MS, the simultaneous detection of aluminium and titanium was proven, for the first time, to be very useful for tracking the spiked titanium oxide particles due to their much higher aluminium content when compared to native titanium dioxide particles in the sunscreen.

This research goes some way to addressing the emerging need for new validated methodology capable of reliably determining size and size-based elemental composition of nanoparticles, and in particular, the development of extraction methods able to preserve the size and composition of nanomaterials in the original sample. This method has been developed further and its feasibility investigated for determining size-based elemental speciation analysis in food.

While robust method development such as this is imperative for characterizing nanoparticle-containing products, the development of standards and reference materials are also essential. They enable standardization and comparability of toxicity tests, and support quality control of existing products.

Reference Materials for Standardization and Comparability

FFF-ICP-MS is used to determine the size distribution and elemental composition of nanoparticles in food and consumer products. Image courtesy of Andrew Brookes <www.andrewbrookes.co.uk>.

The National Measurement Institutes that reside throughout the world produce and distribute high-level measurement standards and measurement procedures, and provide metrological traceability to testing laboratories via their analytical and calibration facilities. LGC, in its designated National Measurement Institute role, is investigating the possibility of developing a standardized panel of reference nanomaterials to enable the development of traceable methods for improved in vitro toxicity measurement for safety assessment.

In vitro screens, which mimic the physiological environment of the human body, offer one of the fastest methods to measure the toxicity of nanoparticles. However, there is poor comparability between laboratories, due, in part, to the fact that when nanomaterials interact with biological systems, their physical and chemical properties can change significantly, affecting their functionality and behavior. For example, nanoparticles tend to become coated with proteins and form agglomerates in biological systems, changing their size and functional properties and altering their behavior under assay conditions. In addition, a lack of standardization in sample preparation methodology and the limited applicability of traditional sizing techniques for heterogeneous nanoparticles in complex matrices further complicate measurements. These issues make prediction of the potential toxicity of nanoparticles difficult. A number of reports6–7 have highlighted the critical need for a bank of nanomaterial reference materials to enable standardized measurement across laboratories.

Currently, there are a limited number of such reference materials available (e.g., 60 nm mono-dispersed gold-citrate [NIST RM-8013]). This inert reference material, although widely used for calibration of equipment, is not best suited for in vitro nanotoxicology due to its lack of toxicity. Recently, under ISO Guide 34 for the production of reference materials, the EU-Joint Research Centre produced a series of nanomaterials in powder-form, characterized for their physical characteristics. These materials are now being used by LGC to form the basis of a standardized panel of prototype reference materials that are stable, biologically active, have reproducible toxicity profiles, and are characterized under biologically relevant conditions.

The research should support public acceptance of nanomaterial safety ...

In order to produce stable reference materials and prevent agglomeration, the behavior of the nanomaterials needs to be controlled by modifying the surface of the particles. These modified particles can then be characterized in biological solutions using instrumentation such as Nanoparticle Tracking Analysis (NTA), which offers a unique method for visualizing and analyzing particle size and size distribution by relating the rate of Brownian motion to particle size. In order to obtain reproducible toxicity profiles, a label-free, real-time, cell-electronic sensing system was used to measure changes in cell number following nanoparticle exposure. This technique enables continual analysis of cells exposed to nanoparticles using electrical impedance measurements and provides quantitative information about the rates and mechanisms of toxicity, which can be missed when using traditional assays. The system is not susceptible to measurement interference by the nanoparticles, thereby allowing the relationship between dose dependant toxic response in the cells and nanoparticle composition to be measured reproducibly. By combining this technique with NTA, nanoparticles in complex suspensions can be characterized in terms of size, distribution, and toxicity.

Once completed, it is anticipated that this research will produce a prototype panel of reference materials characterized for their properties (size, agglomeration state, elemental composition, and toxicity) in physiologically relevant systems so they can be applied as calibration standards in routine testing procedures. The research should support public acceptance of nanomaterial safety by providing reference materials that can be incorporated into testing regimes for regulatory processes.

The development of both methods to measure the size distribution and elemental composition of nanoparticles of relevance to food and consumer products, and the development of characterized reference materials is timely and important for supporting technological and societal challenges associated with sustainable development, competitiveness, food, health, safety, and environmental issues.

Acknowledgement

The work described in this paper is funded by the UK National Measurement System.

References

  1. www.worldmetrologyday.org
  2. Maynard A & Michelson E, The Nanotechnology Consumer Products Inventory. Washington, DC: Project on Emerging Nanotechnologies, Woodrow Wilson International Centre for Scholars, 2011.
  3. Nischwitz V & Goenaga-Infante H, Improved Sample preparation and quality control for the characterisation of titanium dioxide nanoparticles in sunscreens using flow field flow fractionation on-line with inductively coupled plasma mass spectrometry, J. Anal. At. Spectrom., 2012, Advance Article, DOI: 10.1039/C2JA10387G.
  4. Contado C, & Pagnoni A, Commercial sunscreen lotion: flow field-flow fractionation and ICP-AES together for size analysis, Anal. Chem., 2008, 80, 7594-7608.
  5. Samontha A, Shiowatana J, Siripinyanond A, Particle size characterization of titanium dioxide in sunscreen products using sedimentation field-flow fractionation–inductively coupled plasma–mass spectrometry, Anal. Bioanal. Chem., 2011, 399, 973-978.
  6. CELL-PEN: A study to identify the physico-chemical factors controlling the capacity of nanoparticles to penetrate cells, 2009.
  7. EMERGNANO: a review of the environment, health and safety research on nanomaterials and nanotechnology, 2009.

Louise Dean <louise.dean@lgcgroup.com> is working in the Science & Technology group at LGC in Teddington, UK. For technical contacts: Damian Marshall <damian.marshall@lgcgroup.com>, principal scientist for cell biology, or Heidi Goenage-Infante <Heidi.goenage-infante@lgcgroup.com>, principal scientist for elemental analysis. Follow LGC on Twitter: www.twitter.com/LGCGroup.

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