CHARACTERISATION OF NANOPARTICLE SUSPENSIONS BY NTA. SIUPA Agnieszka, HOLE Patrick, WILSON Ian, SMITH Jonathan

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1 CHARACTERISATION OF NANOPARTICLE SUSPENSIONS BY NTA SIUPA Agnieszka, HOLE Patrick, WILSON Ian, SMITH Jonathan NanoSight Ltd, Wiltshire, United Kingdom, EU, Abstract NanoParticle Tracking and Analysis (NTA), has been commercially developed with over 600 systems installed, and is now considered a key characterisation technique in nanomedecine, studying environmental effects on nanoparticles in colloidal suspension and nanometrology. This technique gives significant advantage in sizing over DLS as the individual tracking of particles results in a better ability to measure polydispersed suspension and a better assessment of degree of aggregation. NTA also generates a number-based concentration measurement directly, a crucial parameter in the assessment of dosimetry for nanoparticles where weight-based concentration measurements are less relevant. The sample can have an electric field applied to allow the measurement of zeta potential an important indicator of likelihood of future aggregation. Finally the technique can also be integrated with fluorescence filters to allow fluorescently labelled/loaded particles to be selectively analysed. This can be of particular import when analysing the sample in complex biological media, such as cell culture media or in the presence of protein suspensions. The technique is used for characterisation of exosomes shed from cells for e.g. cell signalling. In this case using NTA in conjunction with fluorescently labelled antibodies may enable speciating the exosomes. Another key application is in measuring a wide range of engineered nanoparticles in more complex media, including river and sea water, cell culture media and buffer solutions. The technique, novel developments and its application to the above fields will be described, explaining the importance of obtaining as complete characterisation as is possible, in as relevant media as possible. Keywords: nanoparticle, sizing, zeta, concentration 1. METHOD Nanoparticle Tracking Analysis (NTA) is a relatively new technique for the characterisation of nanomaterial in suspension. A specially configured laser beam is used to illuminate a small scattering volume within a sample of suspended nanomaterial. The light scattered from each particle is viewed with a conventional optical microscope and video captured with a CCD or CMOS camera. Each individual particle is simultaneously tracked and analysed utilising the Brownian motion to accurately determine the particle size. This technique provides a model-free number based particle size distribution profile. An additional benefit of this particle-byparticle approach is the ability to determine total particle concentration as well as providing concentration data for each size class within the distribution Fig.1 [1].

2 The light scattering properties of nanomaterial can also be used to distinguish between sub-populations within the same sample using the refractive index of nanomaterial Fig.1. When used in fluorescence mode particles can be further differentiated and characterised within complex biological suspensions. Use of green (532nm),blue (488nm) or violet (405nm) lasers, with appropriate filters, allows fluorescent nanoparticles to be seen and tracked. Further information on the material can be obtained through the NTA zeta mode. Applying an electric field across the scattering volume allows nanoparticle electrophoresis to be determined providing information on electrostatic repulsion and therefore colloidal stability of the sample. The minimum detectable nanoparticle size is a function of the particle refractive index but for high Ri materials (e.g. Au) a 10nm minimum threshold is achievable. For lower Ri materials (especially biological) 30-40nm might be the minimum detectable limit. Optimum concentration ranges lie between 10 7 and 10 9 particles/ml. The multiparameter capability of NTA has already been proven as a useful tool in drug delivery [2], protein aggregation studies and viral vaccines [3]. Characterisation of synthesised nanocapsules and particle coatings for targeted drug delivery is an area of high growth for NTA. Additionally, NTA has been used to review drug and protein aggregation and the effect of external factors. This multiparameter approach allows a more complete understanding of nanoscale colloids in the field of biomedicine. Fig.1: Fig A Video of particle under their brownian motions Fig B. Tracked Brownian motion trajectories of nanoparticles as seen by NTA Fig C. 3D plot of 30nm (a) and 60nm (b) gold and 100nm (c) polystyrene 2. CHARCTERISATION OF POLYDISPERSE SAMPLES For the analysis of polydispersed samples (i.e. containing a range of particle sizes) or those which contain different particle types of differing refractive index, the NTA approach is far better suited than more conventional techniques such as Dynamic Light Scattering (DLS) - otherwise known as Photon Correlation Spectroscopy (PCS) due to its particle-by-particle measurement. Because DLS is an ensemble measurement which is significantly biased to larger, higher scattering particles, the resulting intensity weighted average can be seriously misleading in the analysis of polydisperse samples.

3 In a recently published comparison of NTA and DLS, Filipe et al (2010) [4] stated that NTA was shown to accurately analyze the size distribution of monodisperse and polydisperse samples. Sample visualization and individual particle tracking are features that enable a thorough size distribution analysis. The presence of small amounts of large (1,000 nm) particles generally does not compromise the accuracy of NTA measurements and a broad range of population ratios can easily be detected and accurately sized. NTA proved to be suitable to characterize drug delivery nanoparticles and protein aggregates, complementing DLS. They concluded that NTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates. In the following example (Fig.2), also from Filipe et al (2010), the insensitivity of NTA to the presence of contaminants is shown. While present in the scattering volume, the effect they have on the ability of NTA to both see and analyze the smaller particle population is minimal, although high numbers of larger particles may act to occlude smaller particles behind them resulting in a slight loss of accuracy in estimating the number concentration of smaller particles. Fig. 2. Influence of large particles (1,000 nm beads) in a mixture of 100 nm and 400 nm monodisperse beads on NTA and DLS measurements. The size distribution (middle columns) with the corresponding NTA video frame (left columns) and normalized 3D graph (size vs. intensity vs. concentration; right columns) are shown. a) no 1,000 nm beads; b) 1:267 number ratio of 1,000 nm beads to the other beads in the mixture; c) 1:13 number ratio of 1,000 nm beads to the other beads in the mixture 3. Z-NTA Zeta Potential Nanoparticle Tracking Analysis (Z-NTA) adds measurements of electrostatic potential to simultaneous reporting of nanoparticle size, light scattering intensity, fluorescence and count, and does so particle-by-particle. Polydisperse and complex suspensions of both positively and negatively charged particles are readily characterised, and the results are verified by real-time observation of particles moving

4 under both electrophoresis and Brownian motion, without the need for any particle labelling. Changes in the zeta potential distribution with ph, concentration, temperature and particle size can be studied, and aggregation can be measured quantitatively in real-time. The ZetaSight technique allows the simultaneous measurement of size, zeta potential and light scattering intensity for individual nanoparticles in solution. This allows particle populations to be separated in terms of any one of these parameters, and for the relationship between parameters, for example the dependence of zeta potential on particle size, to be studied Fig.3. Fig.3 shows 2-dimensional slices from a 3-dimensional plot, taken from the NanoSight Z-NTA software display, that demonstrates the analysis of two separate particle populations. Results for the 100nm NIST polystyrene size standard in tap water are shown in blue. The white scatter points are for the zeta potential transfer standard (DTS1230), with a size of approximately 300nm.The top panel shows the relationship between light scattering intensity and size, the bottom panel shows the plot rotated to display the relationship between zeta potential and size. 4. APPLICATIONS Given the generic nature of the NTA to be able to detect and analyse particles in the size range nm and analyse them in terms of size, number concentration, refractive index, fluorescence and surface charge, the applications for this technique are widespread in the biological sciences. Accordingly, the technique is used for detecting liposomes, viruses and phage particles in vaccine and drug delivery and gene therapy research. Similarly, the ability to detect particles on an individual basis has proved useful in studies on protein aggregation and product stability in the pharmaceutical sector (FILIPE et al (2010) [4]). In the field of toxicology, the ability to measure nanoparticles and to assess the level of aggregation of nanosuspensions dispersed in various media is critical. NTA is appropriate for rapidly measuring samples as the optimal concentration for detection is very low (10 8 particles/ml) and such samples are commonly polydisperse and has been used extensively to this end [5]. The output, a number-based size distribution and absolute concentration measurements, directly provides the data in the appropriate format. The technique is also used to directly generate a measure of viral concentration. In this regard, the ability of NTA to determine virus count through direct visualization, is of significant value [6]. It has advantage over

5 both plaque assay (measurement times are of just a few minutes and aggregation can be assessed) and qpcr (as all viral particles will be measured whether or not they contain DNA). The application with fluorescence filters to allow fluorescently labelled/loaded particles to be selectively analysed can additionally be of particular import where the suspension is not purified. In the field of pharmaceuticals, a crucial question under inspection is that of protein aggregation and its measurement thereof. In this field NanoSight has been identified as a technique suitable for characterizing this and has crucial benefits over previously available techniques[7]. To date, exosomes research has been constrained by a lack of suitable methods for characterization. NanoSight addresses this need with unique and proven technology. Nanoparticle Tracking Analysis (NTA) allows specific exosomes and microvesicles in the range of nm in liquid suspension to be directly and individually visualized and counted in realtime. Whilst it is frequently adequate to determine merely whether particles of a certain size or size range are present in a sample, it is often much more important to identify and discriminate specific sub-populations of particles within the sample. The NanoSight technique is capable of selectively analysing such populations through, for instance, the use of antibodymediated fluorescent labelling. This approach allows the user to detect, analyse and count only the specific nanoparticles to which the fluorescently-labelled antibody binds, with background non-specific particulates being excluded through the use of appropriate optical filters. Whilst a range of fluorophores can be used, it is advantageous to employ highefficiency, high stability quantum dot labels for best results. Figure 4 shows three size distributions showing: i) all particles present in the STBM sample (blue line) as detected by (non-fluorescent) light scatter; ii) the particles to which the fluorescent QDotlabelled NDOG II antibody had bound specifically, as measured under fluorescence mode (red line) [8]. Engineered Nanoparticles, nanobubbles, inks and pigments can all be characterised as can carbon nanotubes and nanoscale structures (combustion products and contaminants) in solvent based petrochemical and oil sample types. Fig.4 Particle size distributions from scattered light (blue), correct antibody (red) and incorrect (control) antibody (green). Note the number concentration vertical axis. LITERATURE [1] BOB CARR, PATRICK HOLE, ANDREW MALLOY, PHILIP NELSON, MATTHEW WRIGHT, JONATHAN SMITH (2009) Applications of nanoparticle tracking analysis in nanoparticle research - a mini-review, European Journal of Parenteral & Pharmaceutical Sciences 2009; 14(2): [2] [3] [4] VASCO FILIPE, ANDREA HAWE, AND WIM JISKOOT (2010) Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates, Pharmaceutical Research, DOI: /s [5] [6] Anderson, B., et al. Bacteriophage, Volume 1, Issue 2 (2011),

6 [7] Carpenter, J. F., Randolph, T. W., Jiskoot, W., Crommelin, D. J., Middaugh, C. R. and Winter, G. Journal of Pharmaceutical Sciences, (2010), 99: [8] Rebecca A. Dragovic, Christopher Gardiner, Alexandra S. Brooks, Dionne S. Tannetta, David J.P. Ferguson, Patrick Hole, Bob Carr, Christopher W.G. Redman, Adrian L. Harris, Peter J. Dobson, Paul Harrison, Ian L. Sargent Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis. Nanomedicine: Nanotechnology, Biology and Medicine, 7(6), pp. Pages