Enrichment of selective miRNAs in exosomes and delivery of exosomal miRNAs in vitro and in vivo
Enrichment of selective miRNAs in exosomes and delivery of exosomal miRNAs in vitro and in vivo
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DOI：
10.1152/ajplung.00423.2016
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发表时间：
2017-01-01
影响因子：
4.9
通讯作者：
Jin, Yang
中科院分区：
文献类型：
--
作者：
Zhang, Duo;Lee, Heedoo;Jin, Yang
胞外体是由细胞分泌的纳米囊泡，含有多种分子，包括蛋白质、脂肪和DNA/RNA。它们是细胞间通讯的重要媒介，是药物输送和基因治疗的有前途的载体。最近，越来越多的证据表明，microRNAs(MiRNAs)可能成为针对各种人类疾病的治疗干预的新的和潜在的强大靶点。然而，稳定有效地将miRNA模拟物或抑制剂输送到靶细胞仍然是一个主要障碍。为了提高外切递送miRNA分子的效率，开发一种简便有效的方法来富含分离的外切体中的特定miRNAs或反义寡核苷酸是至关重要的。在这里，我们报道了一种新的方法来制备特定的miRNA分子负载的外切体。利用一种改进的氯化钙介导的转染法，我们成功地在分离的外切体中直接增强了指定的miRNA模拟物或抑制物，而不是转染它们的母细胞。我们还将这种方法与电穿孔直接转染外切体的方法进行了比较。这两种方法都证实，外切体可以作为货物，将数量显著增加的选定的miRNA模拟物(S)或抑制物(S)运送到受体细胞。随后，这些miRNA分子丰富的外切体在体内和体外导致指定的miRNAs在受体细胞中高效过表达或缺失。此外，我们证实了外切体递送的miRNA模拟物或抑制物在受体细胞中具有功能。总之，我们开发了一种新的方法来方便高效地操纵外体miRNAs，并成功地将外体miRNA分子运送到受体细胞。
Exosomes are nanovesicles secreted by cells and contain various molecules including protein, lipid, and DNA/ RNA. They are crucial mediators of the intercellular communication and serve as promising vehicles for drug delivery and gene therapy. Recently, accumulating evidence suggests that microRNAs (miRNAs) may serve as new and potentially powerful targets for therapeutic interventions against various human diseases. However, steadily and effectively delivering miRNA mimics or inhibitors to target cells remains a major obstacle. To enhance the efficacy of exosomemediated delivery of miRNA molecules, it is crucial to develop a convenient and efficient method to enrich specific miRNAs or antisense oligos in isolated exosomes. Here we report a novel method to prepare specific miRNA molecule-loaded exosomes. Using a modified calcium chloride-mediated transfection method, we successfully enhanced the designated miRNA mimics or inhibitors in isolated exosomes directly, instead of transfecting their mother cells. We also compared this method with direct transfection of exosomes using electroporation. Both methods confirmed that exosomes can serve as cargos to deliver a robustly increased amount of selected miRNA mimic(s) or inhibitor(s) to the recipient cells. Delivery of these miRNA molecule enriched-exosomes subsequently results in highly efficient overexpression or deletion of the designated miRNAs in the recipient cells both in vivo and in vitro. Additionally, we confirmed that exosome-delivered miRNA mimics or inhibitors are functional in the recipient cells. Collectively, we developed a novel protocol to conveniently manipulate exosomal miRNAs with high efficiency and successfully deliver the exosomal miRNA molecules to recipient cells.