A good judge of character

NanoSight tell us why their characterisation systems provide a unique ability to directly visualise and size nanoparticles in a liquid suspension

THE VISUALISATION process allows each particle to be simultaneously but individually sized overcoming inherent problems associated with techniques such as photon correlation spec¬troscopy (PCS), also known as dynamic light scattering (DLS). Light power scattered from a Rayleigh particle is proportional to sixth power of the radius1 and hence the average particle size produced by techniques such as PCS (which measures fluctuations in intensity of light scattered) is heavily weighted to small numbers of larger contaminant particles. Transmission electron microscopy (TEM) on the other hand requires time consuming sample preparation and imaging and is only able to view a small area, thus risking a non-¬representative analysis of the sample as a whole.

As can be seen in Figure 1, the system may easily distinguish between parti¬cles by the amount of light they scatter. However, particle sizing based on light scat¬tering alone would require knowledge of the re¬fractive index of the particles. The NanoSight technique, known as nanoparticle tracking analysis or NTA, calculates a sphere-equivalent hy¬drodynamic radius based on the Brownian motion of each individual particle and hence is independent of refractive index. The ability to track each particle individually allows better characterization of poly-disperse sys¬tems, whilst the ability to determine the amount of light scattered allows particles of very similar size to be characterised not only by their Brownian motion but also by their relative light scattering ability (figure 2).

As the technique measures the hydrodynamic radius of a particle (i.e. the physical radius of the parti¬cle plus a few nm of a water layer), samples should be prepared in a 1mM salt solution thus reducing the size of the tightly bound water layer surrounding the particle. As the sample measures a hydrodynamic radius, the values measured are a few nanometers larger than as measured in TEM and quoted by the manufac¬turers of the latex standards.

The only preparation required is dilution of the sample to between 10 and 10 particles/ml dependent on sample type and size. At this dilu¬tion, individual particles can be seen moving under Brownian motion and therefore can be analysed. Optimum concentration is particle and solvent dependent.

The importance of particle size in the understanding of wear on orthopaedic implants

As the use of implants made from combinations of polymer, metal and ceramic

Figure 1: Video capture of particles

increases, there is an increasing amount of research being carried out to study the longevity of such devices. Wear caused by friction or corrosion at the articulating surface may generate very small particles on the sub micron scale. It is important to understand how these processes may occur and then to see what affects these nanoparticles may have from a toxicological standing. For example, will these cause an aseptic reaction if they reach certain parts of the body? Will there be any effect on the response of white blood cells?

It has become important to understand not only the size but also the shape and

Figure 2: Tracking individual particles

concentration of such wear-generated nanoparticles.

At the University of Durham, Amy Kinbrum of Professor Tony Unsworth’s group found NTA ideal for the study of metal nanoparticles generated using a pin-on-plate machine to simulate wear. Some metal-metal hip replacements and resurfacings are subjected to different heat treatments while others remain as cast. NTA was ideal for the study of sub micron particles and served to complement the low angle light scattering method used for particles about a micron. The technique is perfect for the study of metal particles as they do not adhere to themselves nor suffer from charging as may be observed in the study of polymeric systems. It was clear that after more cycles, the resultant nanoparticles were of a much narrower and smaller distribution, figures 2 & 3. For each batch of measurements, confirmation of the results was made using TEM.

Dr Joanne Tipper of the Institute of Molecular and Cellular Biology at the

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Figure 3: Distribution curve of polydisperse standards Figure 4: Iker Montes UCD with Nanosight LM10 system

University of Leeds studies nanoparticle sized polymer debris, specifically polyethylene generated first in vitro (to prove its presence) and then in vivo (from tissue from around failed hip replacements). The objective was first to characterize/size the particles and then to consider their bioactivity and effect on cell responses.

Dr Tipper has made measurements on different materials used for implants (metal-metal, ceramic-ceramic and polymer-polymer). She has had good results on model metal and ceramic particle systems. The metal nanoparticle debris are typically in the range of 20-80nm. The NTA results compare extremely well with high resolution FEG-SEM, and these particles compare well with clinically generated wear debris. When studying polymers, NTA produced excellent results for polyethylene particles in the 100-800nm range, again when compared to FEG-SEM.

Particle sizing in nanotoxicology

A new area of research, known as nanotoxicology, has recently emerged with the realisation that particle size can play a key role in a particles ability to infiltrate cells and thus its biological activity.

This has led to an increased need to fully characterise particle size distributions and not just the average size data that some techniques provide. Recent work by graduate student, Iker Montes-Burgos of Professor Kenneth Dawson’s group at University College Dublin has shown the utility of the NTA approach to characterise silica nanoparticles before studying how they enter cells and decrease cell viability. Monomodal particle sizes obtained using NTA broadly agreed with sizes obtained with PCS, (with NTA typically giving a smaller particle size, as expected, due to the intensity weighting of PCS compared to particle-by-particle sizing of NTA). The group is currently trying to correlate how the size and charge of the particles correlates to a reduction of cell viability.

Montes-Burgos has also found that it was possible to measure particle size increase when gold nanoparticles adsorb proteins from complex solutions, such as MEM (minimum essential medium). Particle diameters were shown to increase from 30nm to 40nm on protein adsorption and the work continues with the study of adsorption equilibrium kinetics in different protein solutions including blood plasma.
While a particle may not be toxic in its own right, it may have the ability to absorb and present proteins in altered conformations that transmit unwanted negative effects. Particle size distribution measurements may not be possible using PCS as the technique struggles to differentiate between polydispersed particles. Montes-Burgos concludes that while both PCS and NTA are valid techniques for nanoparticle dispersion analysis, when it comes to polydispersed samples, the NanoSight system delivers more realistic results.