High Throughput and On-Chip Analysis of Engineered Extracellular Nano-Vesicles and Their Functional Characterizations

In recent years, extracellular vesicles (EVs) have shown great promises as drug delivery vehicles to treat various diseases, including cancer and neurological diseases. EV surface engineering is a critical step to achieve targeted drug delivery. By engineering EVs with transmembrane scaffolds, targeting molecules can be displayed on the EV outer surface, and cargos can be loaded into the EV lumen. However, EV heterogeneity and inadequate EV purification and characterization methods impose great challenges on understanding engineered EVs and choosing appropriate transmembrane scaffolds. To this day, surface engineered EVs are still limited to lab research. In this thesis, we modify the EV surface with four transmembrane scaffolds, including native tetraspanins (CD9, CD63 and CD81) and viral envelope glycoprotein (VSVG). We then use a high-throughput and on-chip method to analyze the size and surface marker profile of engineered EVs at single-particle level and measure the engineering efficacy. Our results show that the four transmembrane scaffolds exhibit distinct differences in engineering efficacy and subpopulation integration as well as their impact on EV size and surface marker profile. Consequently, our results provide insights in EV surface engineering by revealing the unique capabilities and characteristics of each transmembrane scaffold. This study may empower the translation of engineered EV from lab research into real clinical applications