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

Abstract

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.

Advances in Exosomes‐Based Drug Delivery Systems

“…Both methods also show differences in the size distribution and surface markers of exosomes. [ 79,80 ]…”

“…In fact, the properties of exosomes depend on their method of isolation. [79,80] Some of the methods implemented achieve high yields but lack specificity while others are very specific but have low yields, as recognized by the MISEV2018 (Minimal information for studies on extracellular vesicles),which uses these criteria for its classification. [81] Differential centrifugation with ultracentrifugation allows for separation based on size, density, and shape.…”

“…[81] Differential centrifugation with ultracentrifugation allows for separation based on size, density, and shape. It consists of successive steps of centrifugation at 4 °C with increasing speed in order to eliminate dead cells, cell debris and contaminating proteins, after which exosomes are obtained via ultracentrifugation at forces ranging from 100 000 × g to 200 000 × g. [79,82,83] This is by far the most frequently used exosome isolation method and it is considered the gold standard to which all other procedures are compared. Indeed, many of the other methods include an initial centrifugation-cleaning step.…”

“…However, it does not work well for highly viscous samples, [84,85] and high centrifugation velocities and times may cause a loss of viable exosomes and some of their components. Moreover, exosomes have low purity, [79] especially when they are obtained from plasma. [84] In order to improve the purification process, additional centrifugation steps [85] or density gradients with iodoxinol o sucrose have been developed.…”

Cross talk between mesenchymal and glioblastoma stem cells: Communication beyond controversies

“…Thus, high performing exosome purification procedures were developed from WJ-MSC to obtain pure populations, a fundamental condition to adopt these vesicles in clinics. 197 Different approaches can be used to modify EV cargo to use them as precision medicine drug carriers, including overexpression of protein of interest in parental cells, antibody or antigen conjugation, chemical modification, or passive and active loading (co-incubation with free drugs, sonication, electroporation, incubation with saponin, antibody binding). 193,198,199 Several approaches were used in this perspective, including EV loading with miRNAs (see previous paragraph) or cytotoxic drugs (doxorubicin, paclitaxel, etc.).…”