Dendrimer-mediated gene delivery: Effect of polymer structure on cellular response
The delivery of nucleic acids to cells, in vitro and in vivo, is becoming a realistic approach for intervention with gene expression at a molecular level. It can be used to repair malfunctioning genes or to introduce new functionalities, with the most important application being clinical treatment of human diseases. The main obstacle on the way to successful gene therapy is to achieve safe and efficient internalization of nucleic acids into the target cells, which requires a carrier that protects the DNA, promotes its interaction with the cell surface, and enables cellular uptake. There is intense research in this field and many new synthetic carriers have been developed. However, there is a lack of systematic studies relating their chemical structure to the resulting transfection efficiency. In this Thesis, we seek to understand factors that are important for successful gene delivery in order to enable improved carrier design. We investigate for a competent class of DNA carriers, cationic dendrimers, how small variations in size and surface structure affect their DNA binding properties and the morphology of the resulting dendrimer-DNA complexes, and in turn what effect this has on cells in vitro. Using advanced spectroscopic techniques we are able to thoroughly characterize the DNA condensates as well as their uptake and subsequent expression in mammalian cells. We have been able to show explicitly, for the first time, that dendrimers bind to DNA in a cooperative manner. We also show that exposure to cell growth media significantly alter dendrimer-DNA complex morphology. These factors are extremely important to consider when trying to relate biophysical studies of dendrimer-DNA complexes to the outcome of transfection experiments. Furthermore, we have developed methods to quantify cellular uptake and to discern different endocytotic mechanisms. These will be applied to the dendrimer library in the near future.