Multi-parameter and Quantitative Characterization of Urinary Extracellular Vesicles at Single-Particle Level

Urinary extracellular vesicles (uEVs) are a rising star in liquid biopsies and have shown great potential in disease diagnosis and treatment monitoring. However, knowledge about the size distribution, particle concentration and biochemical phenotype of uEVs remains poorly understood owing to the limited characterization methods. The large heterogeneity and diversity of EVs call for the development of a high-throughout, multi-parameter technique that can analyze EVs at the single-particle level. Employing strategies for single molecule fluorescence detection in a sheathed flow, our laboratory has successfully developed nano-flow cytometer (nFCM ) that enables detection of single EVs as small as 40 nm. Here, using nFCM, the characterization of uEVs isolated via differential ultracentrifugation was reported. The purity, particle concentration , size distribution , DNA , RNA , and protein expression were reported. The results indicated that the purity of isolated uEVs was about 92% . For uEVs isolated from 10 healthy donors, the concentrations were 3.0 x 10(9)-8.9 x 10(10) particles/mL and the particle sizes fell in the range of 40-120 nm. In addition, DNA and RNA were mainly encapsulated inside the lumen of relatively large-size uEVs and there were no nucleic acids adhered to the surface of uEVs. The percentages of CD9 , CD63 , CD81 or CD24 positive uEVs were also measured. It was demonstrated that nFCM provided an important tool for studying the biochemical properties of uEVs along with their clinical applications.