P11.42 Magnetic targeting of the granzyme B functionalized nanoparticles for therapy of glioblastoma

Abstract BACKGROUND Magnetic drug targeting by employing external magnetic fields represents a promising approach for treatment of glioblastoma. To increase the anti-tumor therapeutic effect magnetic nanocarriers could be functionalized with tumor-targeting bioligands such as granzyme B (GrB). The serine protease GrB that is produced as an effector molecule by activated NK and T cells can induce specific tumor cell apoptosis. MATERIAL AND METHODS The targeting potential of the dextran-coated superparamagnetic iron oxide nanoparticles conjugated with GrB (GrB-SPIONs) was assessed in glioblastoma cells (U87, C6, GL261) by flow cytometry, confocal and electron microscopies. Magnetic field strength (320 mT) in the models of U87 glioblastoma in NMRI nu/nu mice and C6 glioma in Wistar rats was achieved by employing NdFeB cylindrical ferromagnet. The irradiation of the implanted tumors was performed using the CT-image guided Small Animal Radiation Research Platform (SARRP). Accumulation of the nanoparticles was assessed in paraffine-embedded specimens employing Prussian blues staining. Sections were additionally analyzed by IHC for apoptosis (caspase 3). RESULTS Synthesized GrB-SPIONs incorporated into the cytoplasm of tumor cells via the endolysosomal pathway and induced apoptosis in a dose-dependent manner. Intravenous injection of GrB-SPIONs resulted in the glioma-specific retention of the nanoparticles as was shown by high-field (11 T) magnetic resonance imaging and biodistribution studies (NLR-M2 measurements). Magnetic targeting of the nanoparticles in vivo drastically enhanced the accumulation of nanoparticles to the location of the magnet. Thus the local retention of nanoparticles was 12.3-fold higher as compared to application of GrB-SPIONs without external magnetic field. The presence of granzyme B on SPIONs has been shown to promote tumor cells death (IHC staining for caspase 3) and significantly increased overall survival. A combination of nanoparticle treatment with a single radiation dose (10 Gy) significantly prolonged the survival of rats as compared to animals treated only with magnetic targeting of GrB-SPIONs or radiotherapy alone (P<0.001). CONCLUSION Single-agent therapy with GrB-SPIONs demonstrated an impressive increase in overall survival of tumor-bearing animals. Combinatorial regimen employing magnetic targeting and stereotactic radiotherapy further enhanced the therapeutic potential of magnetic conjugates.