Development of EGFR-targeted, drug/dye loaded nanosyringe for selective cellular imaging

Glioblastoma multiforme often has upregulation of extracellular tyrosine kinase receptors such as EGFR and mutant EGFRVIII. This upregulation makes these receptors attractive targets for the binding of drug loaded nanovectors for imaging and patient treatment. We have generated an EGFR-targeted nanosyringe capable of transporting a large drug load to glioma, which undergoes internalization within glioma via the conventional EGF-EGFR coated pit mechanism; PepEGFR-PEG-HCC. The construct is made from an oxidized graphene hydrophobic core functionalized with polyethylene glycol (PEG). Targeting is achieved by ‘clicking’ a EGFR binding peptide on the tips of each PEG, with the peptide identified by phage-display. We have shown that the nanosyringe binds to EGFR, activating the phosphorylation cascade, causing the formation of endosomes via a clathrin mediated endocytosis. This internalization can be halted by EGFR inhibitors such as Erlotinib or by anti-EGFR antibodies. The apparent binding constant for the PepEGFR-PEG-HCC nanosyringe is 20 nM, two orders of magnitude lower than the native peptide, indicating the construct has high avidity. The binding and uptake of the nanosyringe and its drug/dye load is competitive with EGF. High resolution images of U87-EGFR cells exposed to Nile Red-loaded nanosyringes and FITC-labeled EGF show colocalization of both signals. When loaded with the fluorescent chemotherapeutic drug doxorubicin we can observe the real-time binding to gliomal plasma membranes, followed by endosome formation, and then the slow migration of the drug into the nucleus. Intravenous injection of dye loaded nanosyringes into mice with intracranial glioma causes these tumors to take up the nanosyringes and become highly fluorescent. Novel nanosyringes targeting EGFR or other surface epitopes hold great promise for selective cellular imaging of glioblastoma cells. If successful, they may represent a holy grail of imaging, namely selective and imaging of all tumor cells.