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The order of assembly of a magnetic nanoparticle (MNP) vector comprised of the same components (MNP, PEI, and plasmid DNA) on entry mechanism, intracellular localization, and viability of BHK21 cells was investigated. Cellular uptake measurements under four different uptake inhibiting conditions, such as low temperature, depleted cellular ATP, nystatin treatment, and hypertonic environment, show that the cellular entry mechanism of the MNP vector was mediated via clathrin endocytosis. Despite different vector component assembly, all MNP vectors were taken up by the cells through the same mechanism. Labeling and intracellular tracking of the MNP vectors using epi-fluorescence and confocal laser scanning microscopy showed localization of MNP vector within the lysosomes when DNA was assembled on the outer layer of vector. Conversely, when PEI was on the surface of the vector, such that it enclosed both magnetic nanoparticles and the DNA, vector localization in the cell nucleus was observed. The microscopy results demonstrated that the configuration of the MNP vectors dictate the vector's final intracellular target location, and thus the efficiency of transfection. The cellular viability assessment using three different assays further showed that the cellular viability of MNP vector was dose-dependent and varied with the assembly of vector component. All viability assays found negligible toxicity when DNA was on the outer layer of MNP vector except at the highest vector loading. In contrast, attachment of PEI on MNP vector surface induced a significant decrease in cellular viability, due to the ability of PEI on the MNP vector to rupture the lysosomal vesicles.