AbstractsBiology & Animal Science

Targeting mitochondria by mitochondrial fusion, mitochondria-specific peptides and nanotechnology

by Anne Sabine Heller




Institution: Universit├Ąt Regensburg
Department: Chemie und Pharmazie
Degree: PhD
Year: 2013
Record ID: 1099453
Full text PDF: http://epub.uni-regensburg.de/27867


Abstract

This thesis was focused on mitochondria as an intracellular target for drug delivery by the reason that mitochondria are becoming of increasing interest in pharmaceutical and medical research due to their contribution to several diseases (Chapter 1). A protocol for the quick isolation of mitochondria from cultured cells and several methods for the characterization of isolated mitochondria were established. Integrity and functionality of the mitochondrial preparations were demonstrated by a membrane integrity assay, the Cytochrome C Oxidase assay, by staining with a potential sensitive dye, JC-1, and by the analysis of mitochondrial ultrastructure by transmission electron microscopy (Chapter 2). Additionally, a method for the monitoring of long time functionality of isolated mitochondria in terms of their oxygen consumption was established. Typical features of this method included the need of only a few microliters of isolated mitochondria and the possibility of using microplate sensor technology that allowed for the high throughput screening of large sample numbers (Chapter 3). Furthermore, several methods to label isolated mitochondria were investigated. Labeling with fluorescent dyes was a quick and comfortable method but it was not applicable in further studies as the dyes washed out. Therefore, intact cells were transfected with mitochondria targeted green or red fluorescent proteins to label mitochondria permanently and to make them accessible for fluorescence based analytical methods (Chapter 4). To approach the main idea of this thesis, the targeting of mitochondria with nanomaterials by utilization of the mitochondrial fusion process, protocols to accomplish and detect fusion of isolated mitochondria in vitro were established. Mitochondrial fusion in vitro was detected qualitatively by confocal laser scanning microscopy that revealed completely fused mitochondria with mixed matrices due to the merged colors of the fluorescent proteins and by transmission electron microscopy that revealed fusion intermediates of mitochondria with distinct inner membranes and fused outer membranes. Additionally, a protocol for the quantitative evaluation and calculation of mitochondrial fusion efficiency based on flow cytometry was established. Flow cytometry disclosed several advantages such as the capability for high throughput analysis, the possibility to analyze highly diluted samples that did not consume large amounts of isolated mitochondria and an even higher number of mitochondria that could be investigated compared to microscopy techniques that would accompany with time consuming counting of a comparatively low number of mitochondria (Chapter 5). One goal of this thesis was to combine targeting strategies by using specific mitochondrial targeting peptides and nanomaterials, and to utilize mitochondrial fusion to accomplish an uptake of these nanomaterials into mitochondria or to influence on mitochondrial fusion in vitro by these mitochondria targeted nanomaterials. To influence on mitochondrial fusion in vitro…