The prospect of treating debilitating and even fatal diseases by way of genetic-based interventions has been the long-standing goal of gene therapy. However, its widespread clinical use has been severely limited due to a dearth of effective gene delivery systems. Further stalling the development of viable vectors is the lack of clear mechanistic understanding of the barriers that vectors must overcome for successful transfection. The goal of this research is to better understand the structure-function relationships that govern polymer-mediated transfection to serve as guiding principles for the rational design of safer and more efficient polymeric vectors. To achieve this goal, we developed a simple and robust synthetic protocol to synthesize combinatorial libraries of polymeric gene vector candidates. We evaluated three combinatorial libraries of polymeric vectors formulated from single and serially-incremented binary combinations of seven cationic, pH-sensitive, and hydrophobic pendant groups at three different molecular weights (10kDa, 30kDa, 50kDa). High levels of transfection correlated with increasing molecular weights and were achieved by polymers co-functionalized with primary amino and imidazole groups. These high-transfecting polymers appeared to harness the superior charge neutralizing and size condensing capacity of primary amino groups while benefiting from imidazole?s polyplex-stabilizing ability. Imidazole groups were found to effectively bind with DNA via non-electrostatic interactions, resulting in polyplexes that were more resistant to premature polyplex dissociation, which ultimately led to more efficient delivery to cells. The effects on transfection of the polydispersity index (PDI) of polymeric vectors were also evaluated by comparing two identically-formulated libraries, each representing a different PDI (1.3 and 2.0). Within the higher PDI library, we identified several polymer formulations that achieved transfection levels superior to branched-PEI (25kDa), while maintaining superb cell viability. Biophysical characterization of these high-performing polymers revealed a greater degree of polyplex stability compared to their lower PDI, low-transfecting counterparts.
The results from this research have provided a deeper understanding of how several widely used polymeric structures influence gene delivery. The polymeric system and the approach presented herein serves as the foundation upon which future nucleic acid-based therapies (e.g., RNAi therapy) may be based in the move towards safer and more efficient delivery systems.