The two-dimensional (2D) size distributions of DLS and NTA, with the corresponding NTA video frames and three-dimensional (3D) graphs (size vs. intensity vs. concentration) are shown in Figure 4. From these results, the difficulty of DLS in resolving peaks of polydisperse samples becomes apparent, as it was not possible to separate the two bead sizes of any of the mixtures. On the other hand, NTA was able to resolve and distinguish the two size populations in all mixtures and yielded accurate size estimations of the beads in the mixtures. The 2D size distributions show that DLS only gives a single peak for the mixtures shifted towards the larger particle size present, which is again related to its bias to larger particles. The error bars of the DLS results of the two mixtures with the larger bead size (Figure 4c and d) are larger than the ones of the NTA results. This is related to the difficulty that the DLS software has to fit the data of an autocorrelation curve of a sample that has two populations with size differences smaller than the peak resolution limit of this technique. As a result, the single peak as calculated by the DLS software is prone to changes in shape and position from measurement to measurement, giving rise to relatively large error bars in the average result.


Figure 5. Drug delivery nanoparticles measured with NTA and DLS. The size distribution (middle panels) with the corresponding NTA video frame (left panels) and 3D graph (size vs. intensity vs. concentration; right panels) are shown.


The TMC result shows a mean particle size of 320nm by NTA whereas the DLS result is about 410nm. This shift may be explained by the fact that size distributions obtained by DLS are intensity distributions. Because NTA counts each individual particle, it is providing a number distribution.


The PLGA results show a system which is much more polydisperse than the TMC. This is very clearly seen in the visualisation of the sample in the video of the NTA measurement. It is shown that the main population of particles by DLS is shifted to larger sizes than those reported by NTA, which also clearly shows the polydisperse nature of the sample.


In the final liposome example, the DLS result is slightly lower than that obtained using NTA. This may be a function of detection limits of the two techniques and perhaps further analysis by other techniques is required to clarify these observations.


Further examples illustrating the preferred use of NTA to study heat induced protein aggregation have also been published by the Jiskoot group [1] .


Figure 2a. Still image of nanoparticle suspension as seen by microscope in the path of the laser beam; b. trajectories of individual particle Brownian motion as plotted by the tracking analysis program and c. particle size distribution profile as generated by analysis of particle trajectories.


A video of 20-60 seconds is taken of the moving particles at 30 frames per second. The video is analysed by a proprietary analysis program on a frame- by-frame basis, each particle being identified and located automatically and its movement tracked (Figure 2b). The results are finally displayed as a particle size distribution plot (Figure 2c).


A DEMONSTRATION OF THE RESOLUTION OF NTA: COMPARISON OF DATA FROM MIXTURES OF MONODISPERSE POLYSTYRENE BEADS HAVING A FIXED NUMBER RATIO


One of the pitfalls of DLS is its low peak resolution, i.e. it can only resolve particle populations that differ in size at least by a factor of three. Thus, to demonstrate the resolution of NTA, monodisperse polystyrene standard beads were mixed at a fixed number ratio (60nm and 100nm; 100nm and 200nm; 200nm and 400nm; 400nm and 1,000nm) and analysed with both techniques.


CONCLUSION


Figure 4. Size distribution from NTA and DLS measurements of mixtures of monodisperse polystyrene beads (middle panels) with the corresponding NTA video frame (left panels) and 3D graph (size vs. intensity vs. concentration; right panels). a) 60-nm/100-nm beads at a 4:1 number ratio; b) 100-nm/200-nm beads at a 1:1 number ratio; c) 200-nm/400-nm beads at a 2:1 number ratio; d) 400-nm/1,000-nm beads at a 1:1 number ratio.


The two different bead sizes with different scattering intensities can be observed in the NTA video frames and 2D size distribution graphs and can be clearly distinguished in the 3D graphs.


COMPARISON OF RESULTS FROM DIFFERENT DRUG DELIVERY NANOPARTICLES


In order to evaluate the analytical performance of NTA for nanoparticles commonly used in the pharmaceutical field, PLGA (polylactic-co-glycolic acid) particles, TMC (N-trimethyl chitosan) particles and liposomes were analysed with NTA and the results compared to DLS (Figure 5).


The team concluded that NTA has been shown to accurately analyse the size distribution of monodisperse and polydisperse samples. Sample visualisation and individual particle tracking are features that enable a thorough size distribution analysis. The presence of small amounts of large (1,000nm) 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 characterise drug delivery nanoparticles and protein aggregates, complementing DLS.


Commenting on the NTA method, principle user Vasco Filipe, said: “We are able to visualise the sample which gives us confidence in our results. Individual particle tracking enables a much better peak resolution than DLS so making it better suited to study polydisperse samples.”


REFERENCE


[1] NanoSight wishes to acknowledge that the applications shown here along with other examples comparing NTA and DLS were first published in a paper by Vasco Filipe, Andrea Hawe and Wim Jiskoot entitled ‘Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates’. Pharmaceutical Research (2010) DOI: 10.1007/s11095-010-0073-2 (open access at Springerlink.com).


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Figure 3. Professor Wim Jiskoot with Andrea Hawe and Vasco Filipe at Leiden University discuss results from the NanoSight LM-20 system.


Please Contact Gwyneth Astles +44 (0)1727 855574 gwyneth@intlabmate.com


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