Critical evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates

Abstract

Purpose: To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates.

Methods: Standard polystyrene beads of sizes ranging from 60 to 1,000 nm and physical mixtures thereof were analyzed with NTA and DLS. The influence of different ratios of particle populations was tested. Drug delivery nanoparticles and protein aggregates were analyzed by NTA and DLS. Live monitoring of heat-induced protein aggregation was performed with NTA.

Results: 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. Live monitoring of heat-induced protein aggregation provides information about aggregation kinetics and size of submicron aggregates.

Conclusion: NTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates.

Fig. 1

Size distribution from NTA and DLS measurements of monodisperse polystyrene beads. Error bars represent standard deviations obtained from three measurements of the same sample.

Fig. 2

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.

Fig. 3

Influence of different number ratios of 100-nm/400-nm monodisperse beads in NTA and DLS measurements (middle panels) with the corresponding NTA video frame (left panels) and normalized 3D graph (size vs. intensity vs. concentration; right panels).

Fig. 4

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 panels) with the corresponding NTA video frame (left panels) and normalized 3D graph (size vs. intensity vs. concentration; right panels) 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.

Fig. 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.

Fig. 6

IgG aggregates (obtained by heat stress) and insulin aggregates (obtained by metal catalyzed oxidation) 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.

Fig. 7

Live monitoring of IgG aggregation at 50°C in the NanoSight sample chamber. 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.