Evaluation of brain tumor vessels specific contrast agents for glioblastoma imaging

Abstract

A mouse model of glioblastoma multiforme was used to determine the accumulation of a targeted contrast agent in tumor vessels. The contrast agent, consisting of superparamagnetic iron oxide coated with dextran, was functionalized with an anti-insulin-like-growth-factor binding protein 7 (anti-IGFBP7) single domain antibody. The near infrared marker, Cy5.5, was also attached for an in vivo fluorescence study. A 9.4T magnetic resonance imaging (MRI) system was used for in vivo studies on days 10 and 11 following tumor inoculation. T(2) relaxation time was used to measure the accumulation of the contrast agent in the tumor. Changes in tumor to brain contrast because of active targeting were compared with a nontargeted contrast agent. Effective targeting was confirmed with near infrared measurements and fluorescent microscopic analysis. The results showed that there was a statistically significant (P < .01) difference in normalized T(2) between healthy brain and tumor tissue 10 min, 1 h, and 2 h point postinjection of the anti-IGFBP7 single domain antibody targeted and nontargeted iron oxide nanoparticles. A statistical difference remained in animals treated with targeted nanoparticles 24 h postinjection only. The MRI, near infrared imaging, and fluorescent microscopy studies showed corresponding spatial and temporal changes. We concluded that the developed anti-IGFBP7-iron oxide single domain antibody-targeted MRI contrast agent selectively binds to abnormal vessels within a glioblastoma. T(2)-weighted MRI and near infrared imaging are able to detect the targeting effects in brain tumors.

Fig. 1.

A schematic representation of the targeted contrast agent. The contrast agent consists of the superparamagnetic Fe₃O₄ core of mean diameter of 20 nm embedded in a dextran coating (NantoTech Ocean, USA). The core is labelled with infra-red marker (Cy 5.5.) and functionalized with anti-IGFPB7 single domain antibodies.

Fig. 2.

In vivo T₂-weighted MRI of a CD-1 nude brain tumor mouse model. (A) Before, (B) 10 min, (C) 1 h, (D) 2 h, and (E) 24 h post-injection of the targeted (top row) and nontargeted (bottom row) superparamagnetic Fe₃O₄ nanoparticles. A spin echo pulse sequence with the following parameters was used: TR = 5000 ms, TE = 60 ms, FOV = 2 × 2 cm, matrix size 128 × 128, and slice thickness of 1 mm.

Fig. 3.

In vivo MRI of a CD-1 nude brain tumor mouse model. (A) Before, (B) 20 min, (C) 1 h, and (D) 24 h postinjection of targeted (top row) and nontargeted (bottom row) superparamagnetic Fe₃O₄ nanoparticles. A gradient echo flow compensation method was used with the following parameters: FOV = 2 × 2 cm, slice thickness 1 mm, TR = 50 ms, TE = 7 ms, 50 kHz bandwidth, 15 degree flip angle and matrix size 128 × 128.

Fig. 4.

Normalized T₂ relaxation times of tumor and brain measured before and 10 min, 1 h, 2 h, and 24 h postinjection of (A) the targeted and (B) nontargeted NPs.

Fig. 5.

The difference in normalized T₂ relaxation of tumor and brain calculated at time intervals postinjection of the targeted and nontargeted NPs.

Fig. 6.

Targeting of anti-IGFBP7sdAb-targeted Gd-sULVs in an orthotopic brain tumor model. (A) In vivo optical imaging of the head biodistribution of anti-IGFBP7 single domain antibody-targeted (top panels) and nontargeted (bottom panels) iron oxide-Cy5.5 NPs injected in mice bearing orthotopic glioblastoma tumors at various time points (10 min, 2 h, and 24 h). (B) Graph showing changes of the average fluorescence concentration in the brain tumor region in vivo at indicated times after the injection of either anti-IGFBP7 single domain antibody-targeted or nontargeted-NPs-Cy5.5. Data are expressed as mean ± SEM for n = 5 animals. *Indicates significant difference between anti-IGFBP7 single domain antibody-targeted and nontargeted-NP-Cy5.5 (P < .01). (C) Ex-vivo optical imaging of the organ biodistribution of anti-IGFBP7 single domain antibody-targeted (left panel) and nontargeted (right panel) NPs-Cy5.5 24 h after injection in mice bearing orthotopic glioblastoma tumors.

Fig. 7.

Fluorescent microscopic images of mouse glioblastoma multiforme tumor sections obtained 24 h after intravenous injection of anti-IGFBP7 single domain antibody-targeted (A) or nontargeted (B) iron oxide NPs-Cy5.5 (red). Images reveal the strong association of the targeted NP to tumor vessels compared to the nontargeted NP. Mice were also injected with 40 µg of FITC-labeled tomato lectin 10 minutes before sacrifice to stain blood vessels in vivo. Lectin staining (green) co-localizes with the NP-Cy5.5 signal (red) in overlay images to produce yellow-orange. Cell nuclei are stained with DAPI (blue). Scale bar: 100 μm.