Department of Pharmacy-Pharmaceutical Sciences, University of Bari "Aldo Moro", Bari, Italy.
J Control Release. 2013 Dec 28;172(3):1111-25. doi: 10.1016/j.jconrel.2013.09.024. Epub 2013 Oct 2.
Mitochondria represent an attractive subcellular target due to its function particularly important for oxidative damage, calcium metabolism and apoptosis. However, the concept of mitochondrial targeting has been a neglected area so far. The translocator protein (TSPO) represents an interesting subcellular target not only to image disease states overexpressing this protein, but also for a selective mitochondrial drug targeting. Recently, we have delivered in vitro and in vivo small molecule imaging agents into cells overexpressing TSPO by using a family of high-affinity conjugable ligands characterized by 2-phenyl-imidazo[1,2-a]pyridine acetamide structure. As an extension, in the present work we studied the possibility to target and image TSPO with dendrimers. These nano-platforms have unique features, in fact, are prepared with a level of control not reachable with most linear polymers, leading to nearly monodisperse, globular macromolecules with a large number of peripheral groups. As a consequence, they are an ideal delivery vehicle candidate for explicit study of the effects of polymer size, charge, composition, and architecture on biologically relevant properties such as lipid bilayer interactions, cytotoxicity, cellular internalization, and subcellular compartments and organelles interactions. Here, we present the synthesis, characterization, cellular internalization, and mitochondria labeling of a TSPO targeted fourth generation [G(4)-PAMAM] dendrimer nanoparticle labeled with the organic fluorescent dye fluorescein. We comprehensively studied the cellular uptake behavior of these dendrimers, into glioma C6 cell line, under the influence of various endocytosis inhibitors. We found that TSPO targeted-G(4)-PAMAM-FITC dendrimer is quickly taken up by these cells by endocytosis pathways, and moreover specifically targets the mitochondria as evidenced from subcellular fractionation experiments and co-localization studies performed with CAT (Confocal-AFM-TIRF) microscopy.
线粒体是一个有吸引力的亚细胞靶点，因为它的功能对于氧化损伤、钙代谢和细胞凋亡特别重要。然而，到目前为止，线粒体靶向的概念一直是一个被忽视的领域。转位蛋白（TSPO）不仅是一种很有前途的成像靶点，可以对过度表达这种蛋白的疾病状态进行成像，而且也是一种选择性的线粒体药物靶向目标。最近，我们使用一系列具有高亲和力可共轭配体的化合物家族，通过使用 2-苯基-咪唑并[1,2-a]吡啶乙酰胺结构，将小分子的体外和体内成像剂递送到过表达 TSPO 的细胞中。作为扩展，在本工作中，我们研究了用树状大分子靶向和成像 TSPO 的可能性。这些纳米平台具有独特的特性，事实上，它们是用一种比大多数线性聚合物更难达到的控制水平制备的，导致形成具有大量外围基团的近乎单分散的球形大分子。因此，它们是一种理想的递药载体候选物，可以明确研究聚合物的大小、电荷、组成和结构对生物相关性质的影响，如脂质双层相互作用、细胞毒性、细胞内化以及亚细胞区室和细胞器相互作用。在这里，我们提出了一种 TSPO 靶向的第四代[G(4)-PAMAM]树状大分子纳米颗粒的合成、表征、细胞内化和线粒体标记，该纳米颗粒用有机荧光染料荧光素进行标记。我们全面研究了这些树突状聚合物在各种内吞抑制剂的影响下，进入神经胶质瘤 C6 细胞系的细胞摄取行为。我们发现，TSPO 靶向-G(4)-PAMAM-FITC 树突状聚合物通过内吞途径被这些细胞迅速摄取，而且从亚细胞分级分离实验和共定位研究中可以证明，它特别靶向线粒体，这些实验是在共聚焦-AFM-TIRF 显微镜下进行的。
