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Cell-derived exosomes (30-200 nm) as biological "nanocarriers" have attracted a great deal of interest for therapeutic applications due to their ability to internalize in in vivo biological systems (i.e., cells). Although they can be harvested from various sources including stem cells, yet an appropriate isolation and characterization protocol to obtain "pure" exosomal population is needed. For potential clinical applications, understanding the functional ability of exosomes and their purity, that is, free from microvesicles, apoptotic bodies, and protein aggregates, is a pre-requisite. To achieve high purity and yield of exosomes from human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) in the size range of 30-200 nm, we have performed and compared three isolation procedures: ultracentrifugation (UC), sucrose cushion (SC), and commercially available reagent (CR). The isolated exosomes were characterized using nanoparticle tracking analysis (NTA), field emission scanning electron microscopy (FESEM), and atomic force microscopy (AFM). Furthermore, to understand the therapeutic potential of the hWJ-MSC-derived exosomes (hWJ-ME) to target pancreatic tumor cells, the internalization efficacy has been evaluated on the MiaPaCa-2 cell lines using confocal microscopy and flow cytometry. The NTA results showed sucrose cushion to be an optimal method for exosome isolation with high purity (86.8%), as compared to UC (40.5%; p = 0.050) and CR (38%; p = 0.050). Optical analysis by FESEM and AFM revealed that SC-isolated exosomes presented a spherical morphology, whereas UC- and CR-isolated exosomes exhibited an uneven morphology. Furthermore, the data from confocal images and flow cytometry showed that hWJ-ME were internalized by MiaPaCa-2, demonstrating the feasibility of exosomes as a "potential nanocarrier". Thus, our study suggests that a combination of NTA (yield), AFM (dimensions), and FESEM (morphology and topography) could provide sensitive biophysical characterization of hWJ-ME. In the future, enriched exosomes could be used as a delivery vehicle to transport target-specific drugs or gene-silencing constructs to tumors.

Biophysical Characterization and Drug Delivery Potential of Exosomes from Human Wharton’s Jelly-Derived Mesenchymal Stem Cells