EGFRvIII antibody-conjugated iron oxide nanoparticles for magnetic resonance imaging-guided convection-enhanced delivery and targeted therapy of glioblastoma

Abstract

The magnetic nanoparticle has emerged as a potential multifunctional clinical tool that can provide cancer cell detection by magnetic resonance imaging (MRI) contrast enhancement as well as targeted cancer cell therapy. A major barrier in the use of nanotechnology for brain tumor applications is the difficulty in delivering nanoparticles to intracranial tumors. Iron oxide nanoparticles (IONP; 10 nm in core size) conjugated to a purified antibody that selectively binds to the epidermal growth factor receptor (EGFR) deletion mutant (EGFRvIII) present on human glioblastoma multiforme (GBM) cells were used for therapeutic targeting and MRI contrast enhancement of experimental glioblastoma, both in vitro and in vivo, after convection-enhanced delivery (CED). A significant decrease in glioblastoma cell survival was observed after nanoparticle treatment and no toxicity was observed with treatment of human astrocytes (P < 0.001). Lower EGFR phosphorylation was found in glioblastoma cells after EGFRvIIIAb-IONP treatment. Apoptosis was determined to be the mode of cell death after treatment of GBM cells and glioblastoma stem cell-containing neurospheres with EGFRvIIIAb-IONPs. MRI-guided CED of EGFRvIIIAb-IONPs allowed for the initial distribution of magnetic nanoparticles within or adjacent to intracranial human xenograft tumors and continued dispersion days later. A significant increase in animal survival was found after CED of magnetic nanoparticles (P < 0.01) in mice implanted with highly tumorigenic glioblastoma xenografts (U87DeltaEGFRvIII). IONPs conjugated to an antibody specific to the EGFRvIII deletion mutant constitutively expressed by human glioblastoma tumors can provide selective MRI contrast enhancement of tumor cells and targeted therapy of infiltrative glioblastoma cells after CED.

Figure 1. Amphiphilic blocked polymer coated IONPs conjugated to the EGFRvIIIAb

Illustration of IONP (shown in red; core size of 10 nm) coated with a biocompatible amphiphilic copolymer bioconjugated to the EGFRvIIIAb (Illustration provided by Eric Jablonowski, Dept. of Radiology, Emory University School of Medicine). Polyethylene glycols (PEG) are present on the surface of the polymer for further stabilization and biocompatibility of the IONP. Bioconjugation of the EGFRvIII antibody is performed to the –COOH of the polymer coating of the IONP.

Figure 2. Binding of EGFRvIIIAb-IONPs to human glioblastoma cells and MRI contrast enhancement

The binding of EGFRvIIIAb-conjugated IONPs to human GBM cells that overexpress the EGFRvIII mutated protein was confirmed by MRI contrast changes (lower signal intensity at longer TE in T₂-weighted imaging and reduction of T₂ values in Table 1 as shown in B and C after 1 and 2 h of treatment) when compared to the control samples of free IONPs (A).

Figure 3. Cell toxicity analysis of human astrocytes and glioblastoma cells treated with IONPs

A., Human astrocytes were treated with control vehicle (serum-free medium) or IONPs. at 0, 1, 2, and 3 days. No significant toxicity was found with IONP treatment of human astrocytes (P < 0.001). Cell toxicity analysis of human glioblastoma cells (B. U87-MG and C. U87ΔEGFRvIII) after treatment by control vehicle (PBS), IONPs, EGFRvIIIAb, and EGFRvIIIAb-IONPs at 0, 1, and 3 days. A significant decrease in cell survival was found in glioblastoma cells treated by IONPs, EGFRvIIIAb, and EGFRvIIIAb-IONPs at 3 days (P<0.001).

Figure 4. Apoptosis studies and glioblastoma EGFR cellular signaling after treatment with EGFRvIIIAb-IONPs

A., Elevated levels of cleaved caspase-3 and apoptosis were found in all human glioblastoma cells (U87MG, U87ΔEGFRvIII, and U87wtEGFR) after treatment with EGFRvIIIAb-IONPs. B., Elevated levels of cleaved caspase-3 and apoptosis were found in human glioblastoma neurospheres harvested from Patients #74 and 30 after treatment with EGFRvIIIAb-IONPs. C., Glioblastoma EGFR cellular signaling after treatment with IONPs, EGFRvIIIAb-IONPs, control vehicle (PBS), and EGFRvIIIAb. Western blot analysis of glioblastoma cells (U87MG, U87wtEGFR, and U87ΔEGFRvIII) reveals less of the phosphorylated and active form of EGFR (*) in U87ΔEGFRvIII cells after treatment with EGFRvIIIAb-IONPs.

Figure 5. CED of EGFRvIIIAb-IONPs in a mouse glioma model

A. Examples of T₂ weighted images of mouse brains (Mouse 1 and Mouse 2) show the presence of intracranial xenograft (shown by white arrows) and reveal localization, distribution, and dispersion of magnetic nanoparticles days 4, and 7 after CED (shown by black arrows). T₂-weighted MRI showed decrease of signal after CED of IONPs. Areas with signal drop increased 7 days after CED showing dispersion of nanoparticles. B., Initial nanoparticle volume of distribution (V_D) after CED is shown at day 0. The volume of dispersion (V_DI) was determined at 4, 7, and 11 days after CED confirming dispersion of nanoparticles.

Figure 6. Survival studies of athymic nude mice implanted with human U87ΔEGFRvIII xenografts after magnetic nanoparticle CED

A., T₂ weighted MRI showing a tumor xenograft with bright signal 7 days post tumor implantation (arrow); B., Tumor shown (arrow) by contrast enhancement after injection of the gadolinium contrast agent (Gd-DTPA); C., MRI signal drop (arrow) after CED of EGFRvIIIAb-IONPs; D., EGFRvIIIAb-IONP dispersion and T₂ signal drop (arrow) on MRI 4 days after CED. E., Kaplan-Meier survival curve comparison of athymic nude mice after intracranial implantation of human U87ΔEGFRvIII cells and treatment by MRI guided CED of HBSS (control), IONPs, EGFRvIIIAb, or EGFRvIIIAb-IONPs. Statistical significance, P< 0.001, was estimated by log-rank method of CED of EGFRvIIIAb-IONPs, IONPs, and EGFRvIIIAb compared to HBSS CED.