Appropriate surface designs and precise characterizations of nanoparticles (NPs) are important but as of yet unrealized in biomedical applications of nanomaterials. We report a facile synthetic approach to the preparation of iron oxide (Fe3O4) NPs with different surface charges through exchange with polymeric ligands. An accurate analysis method for characterization of the surface-modified Fe3O4 NPs using a combination of the time-of-flight secondary ion mass spectrometry (ToF-SIMS) and Fourier transform infrared spectroscopy (FT-IR) techniques is demonstrated. A comparison of the spatial distribution of the ligand and core particles with ToF-SIMS and FT-IR verified the successful modification and purification. In addition, the surface was shown to play an important role in interactions with live cells in terms of cytotoxicity, cellular uptake, and even gene transfection. Interestingly, precise control over the surface chemistry of NP enabled efficient transfection of siRNA into suspension type cell, the Jurkat human leukemic T cell line, achieving successful down regulation of NOTCH1 whose signaling pathway is known to be involved in acute leukemia. Our report presents an in-depth study of the interactions between the NPs and cells, as well as their synthesis, characterization, and surface-dependent performance properties.