PAMAM dendrimer-based contrast agents for MR imaging of Her-2/neu receptors by a three-step pretargeting approach.

Pretargeting of receptors is a useful approach in molecular imaging and therapy to reduce background noise or toxicity and enhance selectivity. In this study a three-step pretargeting approach that includes a biotinylated antibody, avidin/streptavidin, and a biotinylated imaging agent is described. A PAMAM dendrimer generation 4 (G4D)-based MRI T(1) agent biotin-G4D-DTPA-Gd (bG4D-Gd) and its sister compound with remaining free surface amine groups blocked by succinic anhydride to reduce positive charges (bG4D-Gd-SA) were synthesized. Limited selective enhancement in MRI was observed in a Her-2/neu mouse tumor xenograft by this three-step pretargeting approach that includes biotinylated trastuzumab, avidin and bG4D-Gd, or bG4D-Gd-SA. However, these dendrimer-based MRI agents with molecular weight around 29 kD reached and remained in the tumor through the enhanced permeability and retention effect. Prolonged and extensive accumulation of both bG4D-Gd and b-G4-Gd-SA in the kidneys was also observed.     

Controlled internalization of Her-2/ neu receptors by cross-linking for targeted delivery

Receptor mediated internalization is a crucial step for targeted intracellular delivery of therapeutic and imaging agents. It was recently demonstrated that trastuzumab, an FDA approved humanized monoclonal antibody against Her-2/ neu tyrosine kinase receptor, did not induce endocytosis of the internalization resistant Her-2/ neu receptor. Here we report that accelerated internalization of trastuzumab can be induced by cross-linking the cell membrane bound antibody-receptor complex with an avidin/streptavidin-biotin system. We demonstrated that internalization was achieved both in vitro and in vivo in Her-2/ neu expressing human breast cancer cell lines (BT-474, SK-BR-3 and AU-565) and that repetitive labeling cycles further amplified the loading of cargo molecules within the targeted cells. No trastuzumab binding and internalization was observed in Her-2/ neu negative MDA-MB-231 cells, whereas weak membrane binding and negligible internalization were detected in MCF-7 cells with low expression level of Her-2/ neu receptor. The method was used to noninvasively image Her-2/ neu receptors in isolated cells and in a preclinical breast cancer model with MRI. The controlled internalization of Her-2/ neu receptors can potentially enhance intracellular delivery of drugs and imaging probes, and improve imaging sensitivity and selectivity as well as therapeutic efficacy, through antibody-directed binding and internalization using a pretargeting approach.     

MR molecular imaging of the Her-2/neu receptor in breast cancer cells using targeted iron oxide nanoparticles.

MR molecular imaging is an exciting new frontier in the biomedical applications of MR. One of the clinically relevant targets is the tyrosine kinase Her-2/neu receptor, which has a significant role in staging and treating breast cancer. In this study Her-2/neu receptors were imaged in a panel of breast cancer cells expressing different numbers of the receptors on the cell membrane. Commercially available streptavidin-conjugated superparamagnetic nanoparticles were used as targeted MR contrast agent. The nanoparticles were directed to receptors prelabeled with a biotinylated monoclonal antibody and generated strong T(2) MR contrast in Her-2/neu-expressing cells. The contrast observed in MR images was proportional to the expression level of Her-2/neu receptors determined independently with FACS analysis. In these experiments, iron oxide nanoparticles were attached to the cell surface and were not internalized into the cells, which is a major advantage for in vivo applications of the method.     

Noninvasive detection of temozolomide in brain tumor xenografts by magnetic resonance spectroscopy

Poor drug delivery to brain tumors caused by aberrant tumor vasculature and a partly intact blood-brain barrier (BBB) and blood-brain tumor barrier (BTB) can significantly impair the efficacy of chemotherapy. Determining drug delivery to brain tumors is a challenging problem, and the noninvasive detection of drug directly in the tumor can be critically important for accessing, predicting, and eventually improving effectiveness of therapy. In this study, in vivo magnetic resonance spectroscopy (MRS) was used to detect an anticancer agent, temozolomide (TMZ), in vivo in murine xenotransplants of U87MG human brain cancer. Dynamic magnetic resonance imaging (MRI) with the low-molecular-weight contrast agent, gadolinium diethylenetriaminepentaacetic acid (GdDTPA), was used to evaluate tumor vascular parameters. Carbon-13-labeled TMZ ([¹³C]TMZ, 99%) was intraperitoneally administered at a dose of ∼140 mg/kg (450 mg/m², well within the maximal clinical dose of 1000 mg/m² used in humans) during the course of in vivo MRS experiments. Heteronuclear multiple-quantum coherence (HMQC) MRS of brain tumors was performed before and after i.p. administration of [¹³C]TMZ. Dynamic MRI experiments demonstrated slower recovery of MRI signal following an intravenous bolus injection of GdDTPA, higher vascular flow and volume obtained by T*₂-weighted MRI, as well as enhanced uptake of the contrast agent in the brain tumor compared with normal brain detected by T₁-weighted MRI. These data demonstrate partial breakdown of the BBB/BTB and good vascularization in U87MG xenografts. A [¹³C]TMZ peak was detected at 3.9 ppm by HMQC from a selected volume of about 0.15 cm³ within the brain tumor with HMQC pulse sequences. This study clearly demonstrates the noninvasive detection of [¹³C]TMZ in xenografted U87MG brain tumors with MRS. Noninvasive tracking of antineoplastic agents using MRS can have a significant impact on brain tumor chemotherapy.     

Contributing factors of temozolomide resistance in MCF-7 tumor xenograft models

Vasculature mediated drug resistance in tumors was studied in female SCID mice bearing wild type MCF-7 and adriamycin resistant MCF-7/ADR xenograft using temozolomide (TMZ). A strong tumor growth inhibitory effect of TMZ treatment was observed in MCF-7 tumors during the initial treatment phase with subsequent relapse, but not in MCF-7/ADR tumors. Non-invasive MRI measurements of tumor vascular volume and vascular permeability-surface area product (PS) demonstrated significant reduction of PS in long-term treated MCF-7, but not in MCF-7/ADR tumors. O(6)-Methylguanine-DNA methyltransferase (MGMT) mRNA, and VEGF expression was analyzed using real-time RT-PCR and ELISA, respectively. No significant changes in MGMT mRNA and VEGF expression were observed in either MCF-7 or MCF-7/ADR tumors. However, in vitro incubation of MCF-7 cells with TMZ did induce the expression of MGMT mRNA. In addition, p53 and p21 levels were scored by immunoblotting. Exposure of cells to TMZ did not affect either the p21 or the p53 expression in both MCF-7 and MCF-7/ADR cells. The absence of these molecular responses to TMZ treatment in MCF-7 tumors in vivo supports the possibility that the onset of cancer drug resistance is associated with reduced PS, which can decrease delivery of the drug to cancer cells.     

Noninvasive 1H/13C magnetic resonance spectroscopic imaging of the intratumoral distribution of temozolomide.

Among the primary reasons for failure of anticancer chemotherapy are insufficient drug delivery to the tumor because of inadequate tumor vascularization and/or the antivascular effects of chemotherapy. Thus, determining the spatial intratumoral distribution of anticancer agents by noninvasive methods such as MRI/MRSI is important for monitoring cancer chemotherapy. We therefore studied the distribution of the 13C-labeled anticancer agent temozolomide ([13C]TMZ) in MCF-7 tumor-bearing mice using 1H/13C MRSI. In phantom studies inverse 13C detection with heteronuclear multiple quantum coherence (HMQC) provided a 2.3-fold gain in signal-to-noise ratio (SNR) over direct nuclear overhauser effect (NOE)-enhanced 13C-MRS. This enabled detection of [13C]TMZ in the micromolar range. Three-dimensional (3D) maps of drug distribution with a nominal 2.5-mm isotropic resolution were obtained following intraperitoneal administration of [13C]TMZ, for a total dose of 200 mg/kg. The status of the blood supply of tumors was assessed by gadolinium (Gd)-enhanced dynamic MRI. Nonuniform distributions of the drug and the contrast agent were detected in the tumors. Although carbon-13 MRSI has an inherently low sensitivity for detection, the novel technique described here demonstrates the feasibility of studying the delivery of 13C-labeled drugs and contrast uptake during the course of chemotherapy.