Polymer A-C3, with the second block copolymer of both the ImPAA and BA not only showed the best protection against DNase I with a timed-release mechanism between 24-48 h, but also achieved the highest level of transfection efficiency with 95% of HEK293 cells testing positive for gene expression. To understand the pathways involved in the delivery of pDNA within the cell and the nucleus, several different chemical inhibitors were employed. The addition of the endosome inhibitor, chlorpromazine, resulted in approximately 80% decrease in the number of cells testing positive for gene expression, indicating that the main internalisation pathway into the cell was clathrin-mediated endocytosis. The ability of the polymer/pDNA complexes to escape the endosome was tested through the addition of chloroquine, a chemical known to swell and burst endosomes releasing its contents into the cytosol. Our results show that the polymer A-C3 is efficient at endosomal escape as no increase in transfection efficiency was seen. The final pathway tested was nuclear entry. Nuclear entry of the pDNA is thought to occur either through the nuclear pores or during mitosis when the nuclear membrane is temporarily disintegrated. To determine how the pDNA enters the nucleus, wheat germ agglutinin (WGA), which is known to block the nuclear pores, was added before transfection. Our results show that 48 h post-transfection, transfection efficiency is significantly reduced to less than 5%. We confirmed that reduced transfection efficiency is not due to any toxic effects caused by the WGA. Our results demonstrate that entry occurs primarily though the nuclear pores, and not during mitosis when the nuclear membrane is temporarily disintegrated as the cells would have undergone at least one cell division during the 48 h period.