Two-dimensional (2D) cell models are extensively used in biomedical research to evaluate the efficacy and safety of new drugs. However, these conventional approaches do not precisely mimic the complexity of the organ microenvironment. To overcome this obstacle, three-dimensional (3D) spheroid cell structures usually referred to as spheroids are being developed to better represent the morphological and functional similarity to the tissues. Among several techniques currently employed to produce three-dimensional cell cultures, one of the most promising is the magnetic levitation, which consists of the magnetization of the cells through adsorption of magnetic nanoparticles of iron oxide (Fe3O4), which are produced by the reaction of Fe2+ and Fe3+ ions in alkaline medium. This work produced paramagnetic iron oxide nanoparticles (PIONs) by coprecipitation from an Fe2+ source. The reduction to Fe3+ was obtained by the ionization caused by gamma radiation (60Co) at 15 or 30 kGy radiation absorbed doses. After functionalization with poly-lysine, the nanoparticle suspensions were characterized by XRD, FTIR, zeta potential analysis, DLS and TEM which showed the successful attachment of the carboxylate groups to iron, explaining the ability of the particles to be adsorbed by the membranes. Biological assays showed that these PIONs were biocompatible and efficiently could be applied to develop prostate 3D tumor spheroids model for drug screening.