Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Biochem Biophys Res Commun. 2010 Jun 25;397(2):181-6. doi: 10.1016/j.bbrc.2010.05.070. Epub 2010 May 16.
Mitochondria are the principal producers of energy in cells of higher organisms. It was recently reported that mutations and defects in mitochondrial DNA (mtDNA) are associated with various mitochondrial diseases including a variety of neurodegenerative and neuromuscular diseases. Therefore, an effective mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve this, therapeutic agents need to be delivered into the innermost mitochondrial space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the development of MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. In this study, we provide a demonstration of mitochondrial matrix delivery and the visualization of mitochondrial genes (mtDNA) in living cells using the MITO-Porter. We first prepared MITO-Porter containing encapsulated propidium iodide (PI), a fluorescent dye used to stain nucleic acids to detect mtDNA. We then confirmed the emission of red-fluorescence from PI by conjugation with mtDNA, when the carriers were incubated in the presence of isolated rat liver mitochondria. Finally, intracellular observation by confocal laser scanning microscopy clearly verified that the MITO-Porter delivered PI to the mitochondrial matrix.
线粒体是高等生物细胞中能量的主要产生者。最近有报道称，线粒体 DNA（mtDNA）的突变和缺陷与各种线粒体疾病有关，包括各种神经退行性和神经肌肉疾病。因此，有效的线粒体基因治疗和诊断有望带来巨大的医学益处。为了实现这一目标，治疗剂需要递送到最内部的线粒体空间（线粒体基质），其中包含 mtDNA 池。我们之前报道了一种基于脂质体的载体 MITO-Porter 的开发，该载体通过膜融合将大分子货物导入线粒体。在这项研究中，我们使用 MITO-Porter 展示了在活细胞中进行线粒体基质递药和可视化线粒体基因（mtDNA）的效果。我们首先制备了含有封装的碘化丙啶（PI）的 MITO-Porter，PI 是一种用于染色核酸以检测 mtDNA 的荧光染料。然后，当载体在存在分离的大鼠肝线粒体的情况下孵育时，我们通过与 mtDNA 的缀合证实了 PI 的红色荧光的发射。最后，共聚焦激光扫描显微镜的细胞内观察清楚地证实了 MITO-Porter 将 PI 递送到线粒体基质中。
