Gene therapy is the delivery of therapeutic nucleic acids into a patient's cells in order to prevent or treat genetic disorders or acquired diseases. This is usually achieved by using a gene delivery system as "naked" nucleic acids face many biological barriers on their way to and within the target cells. Most therapies currently on the market are based on viral vectors but due to a number of significant disadvantages, interest has shifted to non-viral alternatives like polycations. These

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... rs are currently the subject of intense research, partly due to their chemical diversity, shelf stability, and diverse architecture. Polyethylenimine (PEI) is often considered to be the gold standard for transfection but suffers from the fact that its transfection efficiency, as well as cytotoxicity, increase with molecular weight. One method to overcome this correlation is to cross-link less toxic, low molecular weight linear PEIs to higher molecular weight branched PEIs with increased transfection efficiency, with reduction sensitive, biodegradable cross-linkers based on disulfides. Numerous studies have demonstrated that the presence of disulfides in polycationic carriers has a favorable influence on many aspects of the gene delivery process. In some cases, however, the presence of disulfides has been shown to have the opposite effect and it is often not well understood why. In fact, many aspects of the delivery are not well understood, partly due to the lack of suitable tools to investigate the delivery process and the carrier degradation. The main goal of this thesis is the development and characterization of new fluorescence-based analytical tools that allow for new insights into the interaction between polycations and nucleic acids, as well as cellular uptake, trafficking, and processing of reduction sensitive polyplexes. This was achieved, among other things, by (1) developing a novel, straightforward method for labeling polyamines with a wide variety of fluorescent probes, including a redox sensitive dye, (2) thor [...]