Performance of Nanoparticle Tracking Analysis in the Measurement of Extracellular Vesicles

Extracellular vesicles (EVs) are membrane-enclosed, non-replicating, submicron-vesicles released by all living cells. Depending on their origin pathway and donor cell, EVs present a specific biomolecular cargo that allows tracing the cell of origin and its status, proposing EVs as potential biomarkers of different diseases, among other clinical applications. Thus, EVs research field is an area of rapid and constant growth. However, the complexity of EVs, based mainly on their heterogeneity in size and cargo, presents unique challenges for their characterization. Nanoparticle Tracking Analysis (NTA) is a technique useful for the quantification and size distribution estimation of nanoparticles in a single particle measurement and is also capable of phenotyping when combined with the fluorescent mode. While the application of NTA in the field of EVs is promising, this technology needs to be improved. On the one hand, the algorithms must be adapted to the heterogeneity of EVs to obtain more accurate measurements. On the other hand, the low refractive index of EVs (a feature with direct impact on the results by optical methods such as NTA) leads to the need of suitable reference materials (RM) for instrument calibration. The aim of this project is to evaluate the performance of NTA for the measurement of heterogeneous populations of EVs. Firstly, SiO2 nanoparticles were used as RM and the standard operating procedures (SOPs) were established for their measurement according to their size. Once established, the SOPs were used for the measurement of different sizes of RM, and EVs derived from cell culture. Our results indicate that to cover the full spectrum of EVs sizes, three SOPs are needed: SOP50 (for the measurements of smaller EVs: 30 - 80nm), SOP100 (80 - 200nm), and SOP500 (200-500). Secondly, the performance of the NTA in fluorescent mode was evaluated for the measurement of synthetic EVs with different fluorophore densities, evidencing that this mode of the instrument depends on the fluorescence intensity regardless of the size of the particle analyzed. Finally, the size of the particles included in this work was measured direct using transmission electron microscopy (TEM). This work details a deep analysis of the performance of NTA, providing valuable data that can be used for the generation of user guidelines for the standardization of EVs characterization. Although more experiments will be necessary to establish determinative conclusions, it is likely that more precise mathematical algorithms are required to truly reflect the heterogeneity of EVs.