Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery.
Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery.
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DOI：
10.1021/acs.molpharmaceut.5b00364
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发表时间：
2015-10-05
影响因子：
4.9
通讯作者：
Jay SM
中科院分区：
文献类型：
--
作者：
Lamichhane TN;Raiker RS;Jay SM
细胞外囊泡（EV）由于其作为促进细胞间分子运输的生物纳米颗粒的性质而具有作为生物治疗剂和药物递送载体的巨大前景。具体而言，EV已被鉴定为核酸的天然载体，引发了对其用于基因治疗和RNA干扰应用的兴趣。到目前为止，小RNA（siRNA和miRNA）已经成功地装载到EV中用于各种递送应用，但是EV用于DNA递送的潜在用途几乎没有被探索。在这里，我们报告了外源线性DNA可以通过电穿孔与EV相关联，其数量足以产生平均每个囊泡数百个DNA分子。我们确定EV中DNA的装载效率和容量取决于DNA大小，与使用该方法的较大线性DNA和质粒DNA相比，长度小于1000 bp的线性DNA分子与EV更有效地结合。我们进一步表明，EV大小也是DNA负载的决定因素，因为较大的微泡比较小的外泌体样EV封装更多的线性和质粒DNA。此外，我们证实了EV通过电穿孔将加载的外源DNA转移到受体细胞中的能力，尽管没有观察到功能性基因递送。这些结果建立了关键参数，告知EV用于基因治疗的潜在用途，并与其他最近的结果一致，表明必须克服实质性障碍，以建立EV作为广泛适用的DNA递送载体。
Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles.