Department of Obstetrics and Gynaecology, University of Auckland, Auckland, 1023 New Zealand.
Department of biomedical Engineering, The Ohio State University, Columbus, Ohio, 43210 United States.
Mol Pharm. 2023 Apr 3;20(4):1898-1913. doi: 10.1021/acs.molpharmaceut.2c00533. Epub 2023 Mar 15.
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.
最近，细胞外囊泡（EVs）作为药物递送的潜在载体，包括基因治疗，引起了相当大的关注。尽管已经研究了来自不同来源的 EV，但该领域目前用于 EV 产生的技术限制了大规模 EV 的生产。胎盘本质上是一种组织移植，具有独特的特性，可以使其避免母体免疫系统的攻击，这使得胎盘 EV 不太可能引发炎症反应，并避免被免疫系统清除。我们提出，从组织培养物中产生的胎盘 EV 是一种生产大量 EV 的有效方法，这些 EV 给药是安全的，并且我们假设胎盘 EV 可以负载大的外源性质粒。为此，我们尝试了三种策略将质粒 DNA 加载到胎盘 EV 中，包括在 EV 分离之前通过电穿孔对胎盘组织进行加载，以及通过电穿孔或直接将 EV 孵育在质粒溶液中来直接加载到胎盘 EV 中。我们报告说，与单层培养的胎盘细胞相比，胎盘释放了大量的 EV。我们展示了在外源加载策略下，大 EV 和小 EV 中都成功地加载了质粒 DNA，并且大 EV 中封装了更多的质粒。重要的是，直接孵育不会改变 EV 的大小或数量。此外，我们表明，EV 中的加载效率取决于外源性质粒 DNA 剂量和 DNA 大小。这些结果使用当前技术提供了对胎盘 EV 中质粒加载能力的现实估计，并展示了胎盘 EV 作为 DNA 递送载体的潜力。
