Nanobioconjugates for Differential Imaging of Solid and Metastatic Brain Tumors

SUMMARY Dynamic contrast enhancement is shown to allow differential MRI detection of human primary brain and metastatic breast cancers in the brain applied to xenogeneic mouse models. Gadolinium-DOTA contrast reagents were covalently attached to polymalic acid (PMLA) nanobiopolymer together with cancer-specific monoclonal antibodies (mAb) previously shown to target primary brain and breast cancer 1-3 . After IV tail injection of tumor-type specific Polycefin TM contrast reagent, enhanced T1 contrast in targeted tumor was sustained for prolonged times, while that in healthy brain or nontargeted tumor was washed out. Differential imaging was confirmed using fluorescence Xenogen imaging. The retention of the specific reagent in the tumor, underlying the mechanism of differential imaging, was demonstrated by confocal microscopy. INTRODUCTION A significant clinical problem with brain metastatic tumors is drug delivery and lack of a specific MRI response identifying the type of cancer at the starting point for optimal treatment. A MRI response in a patient’s brain may result from infection following a preceding chemotherapy, from metastasis of primary lung/breast cancer or from a new primary brain tumor. Unlike lung/breast, brain biopsy is often technically impossible. Here we present an approach to overcome these problems. Figure 1. Schematic presentation of tumor-targeted Polycefin TM contrast reagent for MRI. Gd-DOTA covalently binds to PMLA platform as an amide bond. Maleimide functionalized antibodies and fluorescence dye are attached as thio ethers. EXPERIMENTAL METHODS For generating orthotropic metastatic brain cancers, BT-474 cells for HER-2 positive breast cancer, MDAMB-468 cells for Triple Negative Breast Cancer (TNBC), and U87MG cells for glioblastoma were injected into the brain of mice. After growth, good MRI contrast was acquired with 0.1 mmol/kg of Multihance®, a clinical contrast reagent. For differential imaging with Polycefin TM contrast reagents, the injected amounts were identical and referred to the same value of relaxation T1 as used for Multihance®. Tumor specificity of Polycefin TM contrast reagents was acquired by two types of mAbs attached simultaneously to the polymer (Fig. 1): anti-mouse TfR mAb for extravasation (transcytosis of cancer vessel endothelium) and either Herceptin (targeting HER-2) or Cetuximab (targeting EGFR on TNBC and glioblastoma). Fluorescence labeling with Alexa Fluor 680 was optional for Xenogen IVIS 200 live animal imaging system and confocal microscopy. The synthesis is depicted in Fig. 2. Composition of the contrast reagents in terms of malic acid, mAbs, and gadolinium were quantified post-synthetically. An average amount of 70 attached gadolinium-DOTA per molecule of contrast reagent was synthesized. Relaxivity was 140 mM -1 s -1 per molecule of contrast reagent, the hydrodynamic diameter measured by DLS was 16±2 nm, and of zetapotential -7.0 mV to 8.7 mV, depending on the kind of mAb. For MRI, usually 150 μL solution of contrast reagent having T1 relaxation of 15±1 msec was IV injected via tail. MRI was acquired at Core facility in the Biomedical Imaging Research Institute using a micro MRI 9.4-Tesla Bruker, 94/20 BioSpec MRI system. RESULTS AND DISCUSSION We used the nanobiopolymer platform, PMLA, for synthesis of the contrast reagents to achieve differential brain tumor imaging (Fig. 1). To be independent of EPReffect, extravasation into cancer through transcytosis was targeted by anti-mouse TfR mAb. Specific targeting to cancer cells overexpressing HER-2 or EGFR was achieved by Herceptin and Cetuximab respectively. The contrast reagents were small and nonspherical, not carrying PEG, thus favoring diffusion and moderate circulation times before being washed out from the blood circulation. Figure 2. General scheme for synthesis of Polycefin TM