Validation of Nanoparticle Tracking Analysis in Characterizing Extracellular Vesicle Isolated from Polydisperse Biological Samples

Nanoparticle tracking analysis (NTA) has become a desired method of characterizing extracellular vesicles (EVs) in recent years. The NTA method is distinguished from other EV detection techniques by its ability to simultaneously analyze size and concentration measures in polydisperse EV samples. However, a recent study (Tian et al ., 2016) suggested using a single parametric setting for sizing and counting of polydisperse particles in a broad size range is challenging. The OBJECTIVE of this study is to evaluate the accuracy of the NTA when analyzing polydisperse, biological EV samples isolated at different centrifugation speeds. METHODS Human venous blood samples were collected in a 3.2% buffered sodium citrate‐containing tube. Plasma fraction was separated within 2 hours by a double centrifugation (2,500 g for 15 min each) and stored at −80 °C freezer until analyzed. Microvesicles were isolated from the plasma sample with an ultracentrifugation method using differing speeds (12,000 g , 16,000 g , 20,000 g , and 32,000 g) at 4 °C. Exosomes were isolated by double centrifugation (120,000 g for 70 min each) at 4°C from conditioned media from C2C12 myotubes. Samples of known size microsphere beads with 200, 500, and 800 nm diameters were evaluated in either monodisperse or polydisperse samples for comparison. The NanoSight NS300 was used to measure size distribution and concentration of the particles per manufacturer's procedures. All samples were analyzed using identical camera settings and detection parameters. RESULTS The mean particle size was ranged from 140 nm to 222 nm across the EV samples from human blood. The mean particle size for exosomes from C2C12 cultured media was 204.0 ± 9.1 nm. There was no correlation of mean particle size with centrifugation speed. In all samples, higher concentrations of small particles (< 300 nm) and lower concentrations of larger particles (> 300 nm) were observed. In analyses of sized microsphere samples, monodisperse samples of 200 nm beads accurately measured expected size distribution with a high repeatability. Increases in concentrations by factors of 2 and 4 resulted in accurately measured increases for the 200 nm beads. In contrast, monodisperse samples of 500 and 800 nm beads measured lower concentrations when increased by the same factors. Results for polydisperse samples of 200, 500, and 800 nm beads, with equal concentration ratios (1:1:1) showed lower concentrations for 500 and 800 nm beads, compared to 200 nm (7.29×10 7 , 3.78×10 7 , vs, 10.3×10 7 , respectively). CONCLUSION Regardless of the actual composition of EV subpopulation, NTA measures an average particle size of ~200 nm in polydisperse samples. Analysis of synthetic size beads revealed that this may be because, at least in part, the NTA method underestimates the concentration for larger particles with a size greater than 200 nm. Our results suggest that caution should be used when analyzing polydisperse samples (i.e. serum/plasma) using a nanoparticle tracking method. Support or Funding Information Supported by NIH Grant R01NS102157 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .