DDEL-19PENETRATION OF HOMING PEPTIDE-FUNCTIONALIZED NANOPARTICLES TO GLIOMA SPHEROIDS IN VITRO

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain malignancy. Even when subjected to multimodal treatment using surgery, chemo- and radiotherapy, the majority of GBM patients die within a year after diagnosis. Affinity targeting of anti-glioma drugs to malignant lesions, in particular to disseminated cells hiding behind the blood-brain-barrier, may dramatically improve efficacy of antiglioma therapies. Upon intravenous administration, tumor penetrating peptides (TPP-s) home to malignant lesions and extravasate into tumor parenchyma. Interestingly, these peptides retain their activity to penetrate excised tumor explants in short-term ex vivo assays. Here, we report development of an in vitro 3D penetration assay of homing peptide-functionalized silver nanoparticles (AgNP) in U87 glioma spheroids. U87 spheroids were incubated with fluorescent silver nanoparticles functionalized with prototypic tumor penetrating peptides, iRGD (CRGDKGPDC) and RPARPAR, or control peptides. Subsequently, spheroids were incubated with a non-toxic etching solution that selectively removes the extracellular AgNP with no effect on internalized AgNP. Fluorescence imaging and Matlab -based quantitative analysis of spheroid cryosections and of live spheroids was used to assess distribution of AgNP with and without etching treatment. Compared to control nanoparticles, iRGD and RPARPAR functionalized AgNP showed dramatically increased binding and penetration of the particles in the U87 spheroids. The binding was inhibited by peptide receptor-blocking anti NRP-1 and anti-av-integrin antibodies (but not by control antibodies), suggesting specific effect of AgNP surface peptides. Establishment and validation of 3D in vitro tumor spheroid penetration assay may provide a standardized system for evaluation tumor homing and penetration ability of homing peptides and to allow medium- throughput mechanistic and experimental therapy studies.