From organelle over cell to tissue: Development of fluorescence-based methods to monitor the fate of model transporting structures

The field of nanotherapy engages in the development of nanometer-sized structures that enable or facilitate the transport of active compounds. By using the specific and non-destructive detection mode of fluorescence, this work set out to investigate the fate of fluorescent transporting structures based on small molecules and nanocarriers in biological environments. This was accomplished by the development and application of methods based on Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) which enabled real-time monitoring at a scale below the optical resolution limit of confocal laser scanning microscopy (CLSM). In the first project, the applicability of the small molecular cyanine dyes Cy3 and Cy5 as targeting moieties for the directed transport of reactive groups to mitochondria was investigated. Based on the accumulation of Cy3 and Cy5 in polarized mitochondria, a copper-free click reaction was performed which was traceable by FRET. The reaction yielded a Cy3-Cy5 conjugate with the synergistic property of an enhanced retention in the mitochondria as determined by CLSM. Moreover, the product could not be efficiently imported post-synthetically. This study could hence exemplify the prospect of combining targeted transport and in organello synthesis in order to assemble non-penetrating compounds directly inside mitochondria. A widespread goal of nanocarrier research is to develop transporting structures that remain intact during circulation in the bloodstream but release their payload upon reaching the cell’s interior. The question of studying nanocarrier stability in situ in blood and cells, which suffers from a lack of biocompatible methods, was addressed with FCS. Two independent FCS protocols were developed that enabled a diffusion-based size analysis in the respective biological environment and thus allowed to draw conclusions about the integrity state of the carrier systems. In order to measure nanocarrier stability directly in blood, the second project focused on the development of an FCS method that employed near-infrared excitation and emission. By this, spectroscopic interferences with the high fraction of red blood cells could be minimized. The determination of accurate sizes in the presence of blood cells was furthermore enabled by the application of flow in conjunction with an appropriate analytical fit model. The method’s applicability could be demonstrated on the example of premature dye release from polymeric micelles in blood. In the third project, an FCS protocol on intracellular drug release was established based on the example of fluorescently labeled siRNA that was complexed with cationic lipids (lipoplexes). A great challenge arose from the heterogeneity of the cell’s interior and the simultaneous presence of siRNA complexes and released siRNA. Nonetheless, intracellular FCS measurements yielded a qualitative insight into the incidence of release. Moreover, in a number of cells, which had been transfected either with lipoplexes or nanohydrogel particles and in which the onset of release could not be detected by CLSM, the highly sensitive method of FCS was able to provide evidence for the presence of released siRNA.