| License: Publishing license for publications excluding print on demand (7MB) |
- URN to cite this document:
- urn:nbn:de:bvb:355-epub-278671
- DOI to cite this document:
- 10.5283/epub.27867
| Item type: | Thesis of the University of Regensburg (PhD) |
|---|---|
| Open Access Type: | Primary Publication |
| Date: | 2013 |
| Referee: | Prof. Dr. Achim Göpferich |
| Date of exam: | 8 March 2013 |
| Institutions: | Chemistry and Pharmacy > Institute of Pharmacy > Pharmaceutical Technology (Prof. Göpferich) |
| Keywords: | Mitochondrien, Mitochondrienfusion, isolierte Mitochondrien, intrazelluläres Targeting, Nanotechnologie |
| Dewey Decimal Classification: | 500 Science > 500 Natural sciences & mathematics |
| Status: | Published |
| Refereed: | Yes, this version has been refereed |
| Created at the University of Regensburg: | Yes |
| Item ID: | 27867 |
Abstract (English)
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 ...
Abstract (English)
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 with nanomaterials and mitochondria-specific targeting peptides, several polymers such as 8arm PEGs and a polyetheramine, and nanocarriers such as dendrimers and quantum dots were conjugated to mitochondrial targeting peptides that were known to be recognized by mitochondria, in particular by recognition of the mitochondrial protein import pores. Mitochondrial fusion efficiency in vitro was determined by the previously described protocols based on confocal laser scanning microscopy, transmission electron microscopy and flow cytometry. Thereby, a natural mitochondrial targeting sequence (MLS) and a synthetic mitochondria penetrating peptide (MPP) were investigated. Additionally, mitochondrial fusion was affected unspecifically by highly concentrated polymer solutions. It was shown that mitochondrial fusion could be enhanced by unspecific and specific approaches. PEG 1500 at a concentration of 50 % was the most effective additive for unspecific enhancement of mitochondrial fusion in vitro by dehydration. PEGs with higher molecular weights were not more effective. The MPP modified 8arm PEGs, dendrimers and the polyetheramine Jeffamine® were effective additives for specific enhancement of mitochondrial fusion in vitro. MPP modified 8arm PEGs at a concentration of 1 mM were the most effective enhancer of mitochondrial fusion in vitro. It emerged that carriers with the natural MLS did not enhance mitochondrial fusion efficiency (Chapter 6).
Due to the results of affecting mitochondrial fusion efficiency in vitro, the binding behavior of the mitochondrial targeting peptides MLS and MPP to isolated mitochondria was investigated. Therefore, quantum dots and fluorescent dyes were coupled to the targeting peptides and the binding to isolated mitochondria was determined by flow cytometry. It was not possible to evaluate the binding properties of the mitochondrial targeting peptides MLS and MPP as quantum dots, modified and not modified, bound to cell fragments and isolated mitochondria. Thereby, it was not possible to distinguish between non-specific and specific binding mediated by mitochondrial targeting peptides. The fluorescent dyes TAMRA and BODIPY®, coupled to the MLS, were also not suitable as TAMRA accumulated in mitochondria even without modification due to the positive charge of the molecule and BODIPY® stained isolated mitochondria and cell fragments non-specifically without modification. Therefore, MLS capped gold nanoparticles were synthesized for a transmission electron microscopy study. MLS-modified gold nanoparticles were successfully synthesized by a HEPES reduction method instead of the conventional method of citric acid exchange. The binding of these MLS-capped gold nanoparticles to isolated mitochondria could not be detected in a TEM study. The binding behavior of the MPP was determined by a photoconversion method in transmission electron microscopy using an 8armPEG modified with MPP and the fluorescent dye BODIPY®. The binding of the MPP- and BODIPY®-modified 8arm PEG to isolated mitochondria could also not be detected (Chapter 7).
Even due to the fact that mitochondria are of increasing interest in pharmaceutical and medical research as mitochondrial dysfunction contributes to several severe diseases and even though they exhibit a lot of potential targets, mitochondrial drug delivery and drug targeting is challenging and still at an early stage. Several barriers have to be crossed to achieve a selective targeting and accumulation in mitochondria and there is a need for suitable analytical methods to ensure that applied mitochondrial targeting strategies were successful. Then, it would eventually be possible to verify one goal of this thesis, the uptake of mitochondria targeted nanomaterials into mitochondria by mitochondrial fusion.
Translation of the abstract (German)
Mitochondrien als intrazelluläre Zielstruktur für die gezielte Applikation von Wirkstoffen waren das Kernthema dieser Dissertation. Sie sind an verschiedenen Erkrankungen beteiligt und rücken immer mehr in den Fokus pharmazeutischer und medizinischer Forschungen. Es wurden verschiedene Protokolle etabliert: Zur Isolierung von Mitochondrien aus Zellkultur, zur Charakterisierung isolierter ...
Translation of the abstract (German)
Mitochondrien als intrazelluläre Zielstruktur für die gezielte Applikation von Wirkstoffen waren das Kernthema dieser Dissertation. Sie sind an verschiedenen Erkrankungen beteiligt und rücken immer mehr in den Fokus pharmazeutischer und medizinischer Forschungen. Es wurden verschiedene Protokolle etabliert: Zur Isolierung von Mitochondrien aus Zellkultur, zur Charakterisierung isolierter Mitochondrien hinsichtlich ihrer Ultrastruktur und Funktionalität, zur Mitochondrienfusion in vitro sowie deren qualitative und quantitative Analytik mittels Konfokalmikroskopie, Transmissions-Elektronenmikroskopie und Durchflusszytometrie. Mit den etablierten Methoden wurden eine intakte Ultrastruktur, Funktionalität über mehrere Stunden und Fusionskompetenz gezeigt. Weiterhin wurden verschiedene Methoden zur unspezifischen und spezifischen Beeinflussung der Mitochondrienfusion in vitro mittels hochkonzentrierter Polymerlösungen und Nanocarriern (8arm PEGs, ein Polyetheramin, PAMAM-Dendrimere G4-G7, Quantenpunkte), die mit mitochondrien-spezifischen Peptiden (einer natürlichen mitochondrialen Targetingsequenz, MLS, oder einem Mitochondria-penetrating-peptide, MPP) modifiziert waren, untersucht. Es wurde gezeigt, dass MPP-modifizierte dendritische Polymere die Mitochondrienfusion in vitro erhöhen können. Mitochondrien-spezifisches Targeting ist mit diversen Herausforderungen verbunden und befindet sich einem frühen Stadium wissenschaftlicher Forschungen. Es besteht vor allem ein hoher Bedarf an geeigneten Analysemethoden.
Metadata last modified: 26 Nov 2020 03:01
Owner only: item control page
Download Statistics