Malignant gliomas especially glioblastoma (GBM) are poorly responsive to the 
current treatments. Cilengitide (CGT) is a cyclic pentapeptide that demonstrated 
efficacy for GBM treatment by targeting the integrins avβ3 and avβ5 
over-expressed on GBM cells. However, clinical translation of this therapy has 
been limited by issues including fast blood clearance, high kidney and liver 
uptake, poor blood-brain barrier (BBB) penetration, low tumor specificity and 
rapid washout from tumors. In this study, these issues were tackled in an 
integrated manner using a multi-stage strategy combining ultrasound-targeted 
microbubble destruction (UTMD) with CGT nanotherapy. CGT nanoparticles (CGT-NP) 
prepared using gelatin and Poloxamer 188-grafted heparin copolymer demonstrated 
significant apoptotic and cytotoxic effects in C6 GBM cells. Biodistribution 
study in a rat GBM model demonstrated buildup of high CGT level in tumors 
subjected to CGT-NP+UTMD combined therapy. The tumor CGT level in these animals 
was increased over 3-fold, tumor retention of CGT prolonged and renal clearance 
significantly reduced when compared with free CGT with or without UTMD. 
CGT-NP+UTMD treatment was further shown to extend the median survival period 
from less than 20days in the control and about 30days in free CGT group to about 
80days. This was achieved with low CGT dosing level (2mg/kg twice weekly). In 
situ monitoring of GFAP, Ki67, caspase-3, Beclin-1, and LC-3 in the tumor 
samples together with TUNEL assay, transmission electron microscope imaging and 
Western blot assay all demonstrated high apoptotic and autophagy activities 
induced by the combined therapy. In conclusion, this study has provided 
extensive preclinical data supporting the use of this combined therapy to 
overcome the limitations of standard CGT treatment of gliomas.