Background Magnetic fluid hyperthermia (MFH) relies on the administration to a target tissue of magnetic nanoparticles that generate heat after exposure to an external alternating magnetic field (AMF). The method induces cell death within tumors with the major advantage of minimal invasiveness and deep-seated treatment. Hypothesis We identified two major challenges for the next generation of therapeutics: firstly, a development of new magnetic nanoparticles (MNPs) with a significantly enhanced specific absorption rate (SAR) to maintain a suitable therapeutic temperature inside the tumor cells with minimized dosage; secondly, a controllable and safer way of nanoparticles delivery to deep tumor sites with high selectivity targeting and cellular uptake. Aims The project aims to develop and optimize new shape-anisotropic MNPs that could be efficiently applied for intracellular hyperthermia of prostatic cancer cells (PCa). The focus is to obtain a significant enhancement of SAR of MNPs when are subjected to an AMF, a paramount property to attain the hyperthermic effect at single-cell level with minimized toxicity. We therefore address in vitro and in vivo studies on the interactions of MNPs within the living biological tissue under an applied AMF, and their heat effect at single-cell level. Experimental Design We designed a platform consisting of novel trimagnetic nanoparticles (TMNPs, e.g., ZnFe2O4@Co0.6Zn0.4Fe2O4@MnFe2O4) with controlled shape anisotropy and size distribution for inductive magnetic hyperthermia. The most suitable nanoparticles in terms of highest SAR will be selected for the functionalization with cellspecific targeting molecules to accelerate intracellular accumulation. We will use a PC-3 human metastatic cancer cell line characteristic of prostatic small cell carcinoma. TMNPs will be conjugated with J591 or 5D3 monoclonal antibodies (mAbs) as targeting agent to a transmembrane protein, prostate specific membrane antigen (PSMA). As novelty for a precise quantitative evaluation of the hyperthermia therapy response in vitro and in vivo, we will use a high performance near-field scanning optical microscopy (neaSNOM®) which enables to mapping the cellular receptors and to analysis the proteins in cells structure. SNOM and magnetic romance imaging (MRI) measurements will be carried out to further evaluate the cellular uptake distribution in the treated cells and the efficiency of TMNPs to hyperthermia treatment. Expected Results The general outcome is to create new stimuli-responsive magnetic nanoheaters for efficient intracellular hyperthermia of PCa. We will firstly develop and optimize new superparamagnetic cubic-anisotropic MNPs composed of double magnetic shell of different materials with a significant improvement of the SAR. The second step is related to the functionalization of TMNPs for high selective targeting to the internal structure of PCa cancer cells. Finally, we will focus at obtaining a proof-of-concept for potential applicability of these systems as current therapy (intracellular hyperthermia) in PCa. Impact On Cancer The development of new optimized MNPs as an innovative tool for high intracellular heating performance may lead to significant improvement of current cancer therapy. This novel approach will crucially improve the mechanisms to eliminate suffering and death due to cancer because can be potentially applied nor only to PCa, but to many other kinds of cancer.