--- - |2- Illustration of ACSD nanoparticles (NPs) preparation by the ionic gelation method, and subsequent hypothetical scheme of ACSD NPs internalization. Theranostic nanoparticles (NPs) are promising for opening new windows toward personalized disease management. Using a single particle capable of both diagnosis and drug delivery, is the major benefit of such particles. In the present study, chitosan NPs were used as a dual action carrier for doxorubicin (DOX; chemotherapeutic agent) and superparamagnetic iron oxide nanoparticles (SPIONs; imaging agent). SPIONs and DOX were loaded at different concentrations within poly‐l‐arginine‐chitosan‐triphosphate matrix (ACSD) using the ionic gelation method. NPs’ size were in the range of 184.33 ± 4.4 nm. Drug release analysis of DOX loaded NPs (NP‐DOX) showed burst release at pH 5.5 (as in tumor environment) and slow release at pH 7.4 (physiological condition), demonstrating pH‐sensitive drug release profile. NP‐DOX internalization was confirmed by flowcytometry and fluorescent microscopy. Uptake process results were corroborated by accumulation of drug in the intracellular space. Iron content was evaluated by inductively coupled plasma and prussian blue staining. In vitro magnetic resonance imaging (MRI) showed a decline in T 2 relaxation times by increasing iron concentration. MRI analysis also confirmed uptake of NPs at the optimum concentration in C6 glioma cells. In conclusion, ACSD NPs could be utilized as a promising theranostic formulation for both diagnosis and treatment of glioblastoma. - 'Journal of Cellular Physiology, Volume 234, Issue 2, Page 1547-1559, February 2019. '