Placental Extracellular Vesicles Can Be Loaded with Plasmid DNA.

Recently, extracellular vesicles (EVs) have garnered considerable interest as potential vehicles for drug delivery, including gene therapy. Although EVs from diverse sources have been investigated, current techniques used in the field for EV generation limit large-scale EV production. The placenta is essentially a tissue transplant and has unique properties that allow it to avoid the maternal immune system making it likely that placental EVs will not generate inflammatory responses and will avoid clearance by the immune system. We propose that placental EVs produced from explant cultures are an efficient method to produce considerable quantities of EVs that would be safe to administer, and we hypothesize that placental EVs can be loaded with large exogenous plasmids. To this end, we trialed three strategies to load plasmid DNA into placental EVs, including loading via electroporation of placental tissue prior to EV isolation and loading directly into placental EVs via electroporation or direct incubation of the EVs in plasmid solution. We report that the placenta releases vast quantities of EVs compared to placental cells in monolayer cultures. We show successful loading of plasmid DNA into both large- and small-EVs following both exogenous loading strategies with more plasmid encapsulated in large-EVs. Importantly, direct incubation did not alter EV size nor quantity. Further, we showed that the loading efficiency into EVs was dependent on the exogenous plasmid DNA dose and the DNA size. These results provide realistic estimates of plasmid loading capacity into placental EVs using current technologies and showcase the potential of placental EVs as DNA delivery vehicles.