Amphiphilic surfactants have been used to disperse magnetic nanoparticles in biological media, because they exhibit a dual hydrophobic/hydrophilic affinity that facilitates the formation of a nanoemulsion, within which nanoparticle surfaces can be modified to achieve different physicochemical properties. For the investigation of the interactions of cells with charged magnetic nanoparticles in a biological medium, we selected the nanoemulsion method to prepare water-soluble magnetic nanoparticles using amphiphilic surfactant (polysorbate 80). The hydroxyl groups of polysorbate 80 were modified to carboxyl or amine groups. The chemical structures of carboxylated and aminated polysorbate 80 were confirmed, and water-soluble manganese ferrite nanoparticles (MFNPs) were synthesized with three types of polysorbate 80. Colloidal size, morphology, monodispersity, solubility and T2 relaxivity were found to be similar between the three types of MFNP. However, cationic MFNPs exhibited greater cytotoxicity in macrophages (RAW264.7 cells) and lower cellular membrane effective stiffness than anionic and non-ionic MFNPs. Moreover, cationic MFNPs exhibited large uptake efficiency for RAW264.7 cells compared with anionic or non-ionic MFNPs under the same conditions. Therefore, we propose that surface charge should be a key consideration factor in the design of magnetic nanoparticles for theragnostic applications.