Aim of the present study was to characterize the cellular up-take and the transfection efficiency of BSA-based nanoparticles (NP) in procine brain endothelial cells (PBMEC). NP differed in primary amino-groups (PAG60–147) leading to increased cationization. Cellular uptake was analyzed using confocal laser scanning microscopy. Compared to native BSA (BSA-PAG-60) cellular up-take was enhanced for BSA-PAG95 and BSA-PAG147. Cellular organelle- (Golgi, ER) and membrane-staining were performed to analyze the sub-cellular localization. Colocalization of membrane fractions and NP moving to the perinuclear region were observed by confocal real-time imaging. Accumulation of BSA-PAG95 and BSA-PAG147 in the ER and in the Golgi around the nucleus was observed indicating a retrograde vesicular transport. To identify uptake mechanisms, PBMEC were treated with inhibitors of clathrin- (CLME) and caveolae- (FIL) mediated endocytosis. Uptake of BSA-PAG95 and BSA-PAG147 was significantly reduced by CHLP but not by FIL. Since transport towards the cell nucleus was observed, a GFP-encoding plasmid was coupled to BSA-PAG147 to test whether highly cationized NP can be used for gene delivery. GFP-transfection rate of PBMEC was significantly higher compared to a commercial non-viral vector (Lipofectamine). Our findings demonstrate that modulation of surface charges can turn a native protein into an efficient gene delivery device.