This study describes the synthesis of novel gene delivery vector with low toxicity and high transfection efficiency for magnetofection. The rational design of magnetofection vector called PPMag (PEI-associated polycaprolactone (PCL)-SPIONs) composed of oleic acid (OA) stabilized superparamagnetic iron oxide nanoparticles (SPPIONs) prepared by thermolysis of iron oleate with a combination of hydrophobic PCL and proton absorbing polymer polyethyleneimine (PEI) (PEI-PCL-SPIONs) is described. Encapsulation of amphiphilic PEI with SPIONs not only improves water dispersity of SPIONs, but also allows nucleic acid (NA) condensation and endosomal/lysosomal escape via proton sponge effect after internalization in cells. MTT cytotoxicity assay showed that cell viability was improved compared to conventional PEI-SPIONs. The luciferase activity of magneto-polyplexes treated cells significantly improved compared to both controls revealed that transfection efficiency of PPMag- pCIKlux polyplexes group was improved compared to naked pCIKlux group. The application underneath of a rare earth magnet significantly improve the transfection efficiency (i.e., the luciferase activity doubles) compared to cells without magnet, indicating that sedimentation induced by magnetic field plays important role in accumulation of magneto-polyplexes on cell surfaces. The results demonstrate that PPMag can be used as a novel gene transfection vector to improve transfection efficiency. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 145-154, 2017.