In this application note, we look at how the NanoSight range of instruments uses Nanoparticle Tracking Analysis (NTA) to determine the state of nanoparticles, and in particular their size, size distribution and concentration in test media prior to nanotoxicological and ecotoxicological studies.
Rapid growth in the development and use of engineered nanoparticles (NPs) continues to run ahead of methodologies for the assessment and management of the risks they may pose. Novel properties resulting from particle size at the nanoscale are the basis to the performance promise of nanomaterials. Even where the toxicology surrounding the product in bulk form is well understood, an automatic "read-across" of properties for the same materials at nanoscale is far from an automatic assumption; these are essentially new materials. Awareness grows that the longer term potential toxic effects of such materials and their potential environmental impact are poorly understood. Consequently, there is intense interest and research activity concerning potential toxicological effects of engineered nanomaterials, accompanied by review of the importance of the nanoscale fractions within existing products and formulations.
Before commencing any nanotoxicological study of suspended nanoparticless, it is imperative to know the state of the nanoparticles used, and in particular their size, size distribution and concentration in an appropriate test media.
Particle size determines diffusion rates, penetration of or exclusion by biological barriers and particulate interactions.
This note discusses the application of Nanoparticle Tracking Analysis (NTA) in the characterization of gold nanoparticles and their aggregates in a biologically relevant fluid. Complementary to classical light scattering techniques, NTA allows nanoparticles to be sized and their concentration measured in suspension on a particle-by-particle basis, enabling high resolution understanding of aggregation. This study looks at the change in size of gold nanoparticles, comparing the case of the standard dispersant solvent (citrate buffer) to a dilute plasma (containing proteins), NIST (2007).
Blood was taken from healthy donors. The tubes were centrifuged for 5 minutes at 800 RCF to pellet the red and white blood cells. The supernatant (plasma) was transferred to labeled tubes and stored at -80 ˚C. Upon thawing, the plasma was centrifuged again for 3 min at 16.1 kRCF to further reduce the presence of red and white blood cells. The supernatant was transferred to a new vessel, taking care not to disturb the pellet.
NIST gold standard nanoparticles of 60 nm diameter were used (NIST reference material 8013). These were stored, prepared and used according to the relevant reports of investigation, NIST (2007). The gold was diluted to a concentration of approximately 108 particles/mL using standard citrate buffer with a pH of 7.19. For the dispersions in plasma, the human plasma was diluted 1:106 in citrate buffer, and 10 µL of gold nanoparticles were diluted with 790 µL of the diluted plasma.
Nanoparticle Tracking Analysis was carried out on a NanoSight LM10. All sample preparation and measurements were carried out at University College Dublin, Ireland. Multiple videos, each 166 seconds long, were recorded and analysed in batch mode to ensure statistical robustness. Given that the plasma is a natural ionic medium, it is noted that the issue of protein aggregation will always be problematic.
Videos of the nanoparticles were recorded (Figure 1), tracked and analysed for size and concentration when diluted in citrate buffer and in human plasma (Figure 2).
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The methods for measurement of gold diluted in citrate buffer were as those detailed in the reference materials report, NIST (2007).
NTA can be used to identify both visually and by a concentration measurement that the particles are still monodispersed adding evidence to the hypothesis that proteins in suspension act to coat the nanoparticles (and that the increase in size is not due to aggregation).
The change in size recorded in Figures 1 and 2 show an increase in nanoparticle size of approximately 10 nm, corresponding to a 5 nm thick adsorbed protein layer covering the nanoparticle.
There is a significant and measurable change in particle size distribution of monodisperse gold nanoparticles in the presence of a biological medium such as human plasma. A suitable methodology for the measurement of the thickness of the plasma layer using Nanoparticle Tracking Analysis has been described here.
The breadth and mean of the particle size distribution of the citrate dispersed particles measured by NTA is similar to that determined by NIST. In the presence of plasma, both the particle size and the particle size distribution increase similarly.
NTA offers the ability to measure absolute concentration which may be used to also give a number concentration of the NIST gold nanoparticles. NTA is also ideal for identifying and measuring the concentration of nanoparticle aggregates such as dimers which can be readily differentiated. It thus represents (for the nanoparticles and concentration regimes used here) a useful tool in the existing array of techniques available for bionanoscience and bionanointeractions for engineered nanomaterials.
NTA has been recognized as an analytical method to furnish information about nanoparticle size and, equally importantly, concentration, and is able to do this even in complex sample types of high polydispersity (Montes-Burgos et al., 2010; Lynch, 2008; Montes-Burgos et al., 2007). Methods such as NTA can be considered as one of a number of means by which the environmental impact and potential cellular toxicity of nanoparticles could be studied in the future (Borm et al., 2006; Tenuta, 2008; Tran and Anton, 2009; Kuhlbusch et al., 2010; Hassellöv and Kaegi, 2009; Stolpe et al., 2011).
Recent studies have shown that the complexity of interactions between NPs and environmental matrices is extremely complex and represents a significant challenge in both their quantification and modelling but in which NTA may provide a mechanism of investigation and analysis (Gornati et al., 2009; Hartmann, 2011; Arvidsson et al., 2011; Howard, 2010; Njuguna et al., 2011; Tran et al., 2009).
In a study of the effects of a coating applied to zero-valent nano-iron (nZVI) on early life stage development of three key marine invertebrate species, Kadar et al. (2012) used NTA to study the dissolution of nZVI in seawater showing that the coating helped stabilize the nanometal suspension. Kadar has also studied the effect of NTA-analyzed industrially relevant engineered iron nanoparticles on growth and metabolic status of marine microalgae cultures in which he described subsequent alterations in their growth rate, size distribution, lipid profiles and cellular ultrastructure (Kadar et al., 2012)
Using a 15k oligonucleotide microarray for Daphnia magna, a freshwater crustacean and common indicator species for toxicity, to differentiate between particle specific and ionic silver toxicity and to develop exposure biomarkers for citrate-coated and polyvinylpyrrolidone-coated silver NPs, Poynton et al. (2012) determined the degree of aggregation of silver NPs by NTA prior to studying their toxicity at the genomic level.
In studying the toxicity of zinc oxide nanoparticles to Folsomia candida, Waalewijn-Kool et al. (2012) showed that differences in methods of spiking exposure media to test dispersion size characteristics made little difference to the reproductive capacity of the organism, NTA and TEM both showing the toxicity of the zinc oxide was not related to particle size.
At a cellular level, NTA has proved useful in studying the genotoxicity of cobalt NPs in human peripheral leukocytes (Colognato et al., 2008) and mouse fibroblasts (Ponti et al., 2009). The ability of nanoparticles to cross the human placenta (Wick et al., 2009) and methods to study their effects on other biological barriers have been addressed (Linn et al., 2010) including the transport of silicon dioxide nanoparticles through human skin (Staroňová et al., 2012). Similarly, Filon et al. (2012) reported on human skin penetration of cobalt nanoparticles through intact and damaged skin, suggesting that Co applied as NPs is able to penetrate the human skin in an in vitro diffusion system.
An understanding of the distribution of nanoparticle sizes prior to their introduction to cellular systems for cytotoxological testing is crucial and NTA has proved useful in this regard (Kendall et al., 2009; Patel et al., 2010; Munaro, 2010; Karlsson, 2010). The chemical interactions of nanoparticles of different types with various matrices of biological origin such as serum (Treuel et al., 2010) and organic pollutants (Ben-Moshe et al., 2009) and dithiothreitol (Sauvain et al., 2008) have also been studied.
The toxicological effects of cobalt nanoparticles (Co-NPs) aggregates were examined and compared to those of cobalt ions using different cell lines representing lung, liver, kidney, intestine and the immune system. The overall findings were in line with the hypothesis that the toxic effects of aggregated cobalt NPs are mainly due to cobalt ion dissolution from the aggregated NPs. (Limor et al., 2011).
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