Lange Tim, Maron Luzia, Weber Claudia, Biedenweg Doreen, Schlüter Rabea, Endlich Nicole
Institute of Anatomy and Cell Biology, University Medicine Greifswald, Friedrich-Loeffler-Str. 23c, 17487, Greifswald, Germany.
Institute for Physics, University of Greifswald, Greifswald, Germany.
J Nanobiotechnology. 2025 May 23;23(1):373. doi: 10.1186/s12951-025-03426-7.
Podocytes are a crucial component of the glomerular filtration barrier, and changes in their 3D structure contribute to over 80% of chronic kidney disease (CKD) cases. Exosomal small RNAs play a key role in cell-cell communication in CKD and may serve as nanocarriers for delivering small RNAs into podocytes. However, the uptake of exosomal cargo by podocytes remains poorly understood. This study explores the use of isolated exosomes, directly transfected with fluorescently-labeled small RNAs, for tracking and delivering small RNAs to cultured podocytes.
Exosomes were isolated from immortalized murine podocytes and transfected with Cy3-labeled siRNA and miRNA controls using the ExoFect siRNA/miRNA Transfection Kit. We characterized the transfected exosomes via transmission electron microscopy (TEM) and Western blot for exosomal markers CD9 and TSG101. Subsequently, we co-cultured these exosomes with podocytes and used confocal laser-scanning microscopy (cLSM), and structured illumination microscopy (SIM) to visualize cargo uptake, confirmed through flow cytometry, imaging flow cytometry and immunofluorescence staining for Rab5, Rab7, and CD9. The isolated exosomes were also transfected with pre-miR-21 and filamin A (FlnA)-siRNAs before being co-cultured with podocytes. We confirmed the efficiency of transfection and knockdown using RT-qPCR, Western blotting, and immunofluorescence staining.
TEM revealed that the exosomes maintained a consistent shape and size of approximately 20 nm posttransfection and exhibited a stable expression of CD9 and TSG101. Flow cytometry and immunofluorescence imaging showed that podocytes take up Cy3-labeled exosomal miRNAs and siRNAs time-dependently, utilizing various mechanisms, including encapsulation within vesicular structures, endocytosis and free distribution within the cells. Transfection of exosomes with FlnA-siRNAs resulted in a significant 2.8-fold reduction of filamin A expression in co-cultured podocytes, while pre-miR-21-transfected exosomes led to a remarkable 338-fold increase in mature miR-21 levels.
These findings demonstrate that direct exosome transfection with fluorescently-labeled small RNAs is an effective method for tracking exosomal cargo in podocytes. This study is the first to show that directly transfected exosomes can deliver small RNAs to podocytes in vitro, suggesting their potential as RNA carriers for therapeutic strategies in more complex settings.
足细胞是肾小球滤过屏障的关键组成部分，其三维结构的改变导致超过80%的慢性肾脏病（CKD）病例。外泌体小RNA在CKD的细胞间通讯中起关键作用，并且可能作为纳米载体将小RNA递送至足细胞。然而，足细胞对外泌体货物的摄取仍知之甚少。本研究探索使用直接用荧光标记的小RNA转染的分离外泌体，用于追踪小RNA并将其递送至培养的足细胞。
从永生化小鼠足细胞中分离外泌体，并使用ExoFect siRNA/miRNA转染试剂盒用Cy3标记的siRNA和miRNA对照进行转染。我们通过透射电子显微镜（TEM）和针对外泌体标志物CD9和TSG101的蛋白质印迹对外泌体进行了表征。随后，我们将这些外泌体与足细胞共培养，并使用共聚焦激光扫描显微镜（cLSM）和结构照明显微镜（SIM）来可视化货物摄取，通过流式细胞术、成像流式细胞术以及针对Rab5、Rab7和CD9的免疫荧光染色进行确认。分离的外泌体在与足细胞共培养之前也用pre-miR-21和细丝蛋白A（FlnA）-siRNAs进行了转染。我们使用逆转录定量聚合酶链反应（RT-qPCR）、蛋白质印迹和免疫荧光染色确认了转染和敲低的效率。
TEM显示，转染后的外泌体保持一致的形状和大小，约为20nm，并表现出CD9和TSG101的稳定表达。流式细胞术和免疫荧光成像显示，足细胞以时间依赖性方式摄取Cy3标记的外泌体miRNAs和siRNAs，利用多种机制，包括包裹在囊泡结构内、内吞作用以及在细胞内自由分布。用FlnA-siRNAs转染外泌体导致共培养的足细胞中细丝蛋白A表达显著降低2.8倍，而用pre-miR-21转染的外泌体导致成熟miR-21水平显著增加338倍。
这些发现表明，用荧光标记的小RNA直接转染外泌体是追踪足细胞中外泌体货物的有效方法。本研究首次表明，直接转染的外泌体可以在体外将小RNA递送至足细胞，表明它们作为更复杂环境中治疗策略的RNA载体的潜力。
