Imaging flow cytometry (IFC) is a form of high-throughput microscopy which combines the advantages of fluorescent microscopy with the quantitative capabilities of traditional flow cytometry. This platform has recently been standardized for quantitatively analyzing the cellular uptake of extracellular vesicles (EVs), which are nanosized lipid bilayer-bound vesicles that are naturally secreted from most cell types as a communication mechanism to deliver proteins, lipids, and genetic material. Due to their innate characteristics and low immunogenicity and toxicity, EVs are used as therapeutic delivery shuttles when loaded with a cargo. However, there are limited in vitro systems with quantitative methods for use in selecting optimal EV source(s) or how loaded cargo and other modifications alter cellular uptake efficiency and target specificity. Here we further explore IFC as a platform for characterizing cellular uptake of neural stem cell-derived extracellular vesicles (NSCEVs) by neurological disease-relevant in vitro cell systems, including human mature neurons and a neurovascular cell line mimicking the blood brain barrier (BBB) which prevents the entry of majority of therapeutics into the central nervous system. We found that NSCEVs are efficiently internalized by both neural cells in vitro. The quantified results from IFC suggest that NSCEVs were internalized at a higher quantity by neural cells comparing to HEK293T cells. These data support the use of NSCEVs as a promising therapeutic shuttle to overcome the BBB and enhance the drug delivery and target efficiency for neurological disorders. The IFC-based method we developed to quantitatively characterize the cellular uptake and specificity of nanodelivery shuttles in a high throughput manner will help the selection and optimization of EVs for specific diseases and facilitate EV-based therapeutics.

VExtracellular vesicles, Cellular uptake, Neurological disorders, Imaging flow cytometry, Therapeutic delivery