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Gene therapy holds great potential to enhance human medicine since it could provide treatment to a vast variety of diseases. Viral gene delivery vectors have high transfection efficiency, but often they are associated with high risk of immunogenicity as well. Non-viral vectors, especially polycationic polymer based gene carriers provide the advantages of low immunogenicity and ease of synthesis, but they face two major challenges for a broader application in clinical trials: high toxicity and low transfection efficiency. In this study, we performed experiments to gain a better understanding of the mechanisms and pathways underlying the high toxicity and low transfection efficiency associated with polyplex-mediated gene delivery. In the first part of the thesis, we studied the toxicity resulted from polyplex-mediated gene delivery and found that polyplexes made from PEI/DNA plasmids were able to induce autophagy in fibroblasts, a third type of cytotoxicity besides apoptosis and necrosis. With the proper choice of treatment time and drug concentrations, we decoupled cell cytotoxicity due to apoptosis and necrosis from autophagy, and found that transfection efficiency is positively correlated with the regulation of autophagy. In the second part of the thesis, we performed in vitro physical characterization of polyplexes formed from six different polymers in order to understand their local in vivo performance. Homopolymer with a larger molecular weight (and polymer chain) was able to transfect cells in vitro, but not in vivo. On the other hand, a shorter polymer chain favored local in vivo transfection. PEGylation helped the polyplexes to be stable against serum protein, but PEGylation of long chain homopolymer did not improve its in vivo performance. Taken together, these findings could help us to improve transfection efficiency through modulation of cytotoxicity (autophagy) and gain a better understanding of polyplex in vivo performance based on its in vitro behavior and therefore providing insight into the design of gene delivery vehicle.