A multifunctional nanoprobe capable of targeting glioma cells, detectable by both magnetic resonance imaging and fluorescence microscopy, was developed. The nanoprobe was synthesized by coating iron oxide nanoparticles with covalently bound bifunctional poly(ethylene glycol) (PEG) polymer, which were subsequently functionalized with chlorotoxin and the near-infrared fluorescing molecule Cy5.5. Both MR imaging and fluorescence microscopy showed significant preferential uptake of the nanoparticle conjugates by glioma cells. Such a nanoprobe could potentially be used to image resections of glioma brain tumors in real time and to correlate preoperative diagnostic images with intraoperative pathology at cellular-level resolution.

Nanoparticle-based platforms have drawn considerable attention for their potential effect on oncology and other biomedical fields. However, their in vivo application is challenged by insufficient accumulation and retention within tumors due to limited specificity to the target, and an inability to traverse biological barriers. Here, we present a nanoprobe that shows an ability to cross the blood-brain barrier and specifically target brain tumors in a genetically engineered mouse model, as established through in vivo magnetic resonance and biophotonic imaging, and histologic and biodistribution analyses. The nanoprobe is comprised of an iron oxide nanoparticle coated with biocompatible polyethylene glycol-grafted chitosan copolymer, to which a tumor-targeting agent, chlorotoxin, and a near-IR fluorophore are conjugated. The nanoprobe shows an innocuous toxicity profile and sustained retention in tumors. With the versatile affinity of the targeting ligand and the flexible conjugation chemistry for alternative diagnostic and therapeutic agents, this nanoparticle platform can be potentially used for the diagnosis and treatment of a variety of tumor types. [Cancer Res 2009;69(15):6200-7]

Converging advances in the development of nanoparticle-based imaging probes and improved understanding of the molecular biology of brain tumors offer the potential to provide physicians with new tools for the diagnosis and treatment of these deadly diseases. However, the effectiveness of promising nanoparticle technologies is currently limited by insufficient accumulation of these contrast agents within tumors. Here a biocompatible nanoprobe composed of a poly(ethylene glycol) (PEG) coated iron oxide nanoparticle that is capable of specifically targeting glioma tumors via the surface-bound targeting peptide, chlorotoxin (CTX), is presented. The preferential accumulation of the nanoprobe within gliomas and subsequent magnetic resonance imaging (MRI) contrast enhancement are demonstrated in vitro in 9L cells and in vivo in tumors of a xenograft mouse model. TEM imaging reveals that the nanoprobes are internalized into the cytoplasm of 9L cells and histological analysis of selected tissues indicates that there are no acute toxic effects of these nanoprobes. High targeting specificity and benign biological response establish this nanoprobe as a potential platform to aid in the diagnosis and treatment of gliomas and other tumors of neuroectodermal origin.

Aims-This study examines the capabilities of an actively targeting superparamagnetic nanoparticle to specifically deliver therapeutic and magnetic resonance imaging contrast agents to cancer cells. Materials & methods-Ironoxide nanoparticles were synthesized and conjugated to both a chemotherapeutic agent, methotrexate, and a targeting ligand, chlorotoxin, through a poly(ethylene glycol) linker. Cytotoxicity of this nanoparticle conjugate was evaluated by Alamar Blue cell viability assays, while tumor cell specificity was examined in vitro and in vivo by magnetic resonance imaging.Results & discussion-Characterization of these multifunctional nanoparticles confirms the successful attachment of both drug and targeting ligands. The targeting nanoparticle demonstrated preferential accumulation and increased cytotoxicity in tumor cells. Furthermore, prolonged retention of these nanoparticles was observed within tumors in vivo.Conclusion-The improved specificity, extended particle retention, and increased cytotoxicity toward tumor cells demonstrated by this multifunctional nanoparticle system suggest that it possesses potential for applications in cancer diagnosis and treatment.Keywords iron oxide; nanoparticle; chlorotoxin; methotrexate; tumor; drug delivery; magnetic resonance imaging The development of tumor specific multifunctional nanoparticles is currently an area of intense research with the potential to revolutionize the diagnosis and treatment of cancer. These particle systems, also referred to as nanovectors, have been envisioned as novel contrast agents for non-invasive molecular imaging and targeted carriers for drug delivery [1,2]. As a major class of these materials, superparamagnetic iron oxide nanoparticles have been examined extensively for applications in cancer diagnosis and therapeutics due to their biocompatibility and magnetic properties [3,4]. Early forms of these nanoparticles, which exploit the body's natural clearance pathways (e.g. reticuloendothelial system (RES)-mediated passive targeting), * Author for correspondence: Department of Materials Science & Engineering, 302L Roberts Hall, University of Washington, Seattle, WA 98195-2120, Tel: (206) 616 9356, Fax: (206) 543 3100, mzhang@u.washington.edu. NIH Public AccessAuthor Manuscript Nanomedicine (Lond) In addition, the magnetic properties of these iron oxide nanoparticles have also been examined as a means of remotely directing therapeutic agents specifically to a disease site [7]. Nanoparticles possess a high surface area-to-volume ratio allowing them to be loaded with large amounts of a therapeutic or cytotoxic drug. As with other drug delivery systems, specific accumulation in malignant tissues results in reduced dosages necessary for a therapeutic effect and less deleterious side-effects associated with non-specific uptake of cytotoxic drugs by healthy tissue. Additional advantages of drug carrier systems over traditional systemic chemotherapy include the ability to improve the solubility of hydrophobic drugs, stabilizatio...

ABSTRACT9L rat glioma cells have been used as a model for brain tumor therapies. It has been reported that in vivo infection of 9L cells with a replication-defective retrovirus expressing the herpes simplex thymidine kinase gene resulted in decreased tumor formation following treatment with the antiviral drug ganciclonr. the study reported here, rats were injected either intracerebrally or subcutaneously with 9L glioma cells expressing a chimeric hygromycin phosphotransferase-thymidine kinase fusion protein or with unmodified 9L cells. Tumor formation was decreased in the rats receiving modified cells, even in the absence of treatment with gacc vir. Suppression of tumor growth was also observed with cells modified to express the intracellular selectable marker neomycin phosphotransferase. These results indicate that an intracellular selectable marker, in the absence of pharmacologic selection, can inhibit tumor growth of 9L cells. The demonstration that intracellular marker genes can negatively influence the survival of transplanited cells has important implications for in vivo studies that use genetically modified cells.The median survival ofpatients with a malignant glioma is x1 year following diagnosis, even with aggressive surgery, radiation therapy, and chemotherapy (1). New treatment regimens for gliomas are frequently tested in animal models, often using the 9L rat glioma cell line. Several reports have used retroviral vectors to demonstrate in vivo transduction of C6 or 9L gliosarcoma tumors (2-5). Culver et al. (2) reported the effective treatment of an experimentally implanted 9L rat glioma by infection with a retrovirus conferring sensitivity to the drug ganciclovir. The investigators demonstrated that intracranially implanted 9L cells could be infected with a replication-defective retrovirus expressing the herpes simplex virus thymidine kinase (tk) gene following injection of the tumor with the viral packaging cell line. All animals were treated with ganciclovir, and 11 of 14 animals that received the virus-producing packaging cell line did not develop tumors in the 15-day observation period prior to sacrifice. Subsequent studies extended these findings and demonstrated that ganciclovir treatment was necessary for the prevention of tumor formation during the first 3 weeks after cell injection (3). These results and related studies (4-7) have provided the basis for human trials in which intratumor injection of a packaging cell line that produces an amphotropic retrovirus expressing the tk gene is followed by treatment with ganciclovir (8). We have attempted to reproduce the findings of Culver et al. (2)

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Medulloblastoma, an invasive embryonal tumor of the cerebellum, is the most common malignant brain tumor of childhood. Genome-wide technologies have recently shown that gene-specific changes in DNA methylation status are a significant feature of medulloblastoma development. Mouse models of medulloblastoma may provide important systems in which to study the impact of these DNA methylation changes, however the epigenetic basis of these models has not been widely investigated. We therefore adopted a cross-species approach to identify epigenetic events of relevance in human and mouse tumors, and investigate their impact on medulloblastoma development. Using Illumina Goldengate DNA methylation arrays, we first identified a series of tumor-specific DNA methylation changes (measured relative to the normal cerebellum) which are strongly associated with activation of the Sonic hedgehog (SHH) signalling pathway, which occurs in approximately 25% of human primary tumors. From these, 12 candidate CpG sites, which lay within regions orthologous to the mouse genome, were selected for further investigation, alongside RASSF1A and COL1A2 CpG sites, which show frequent methylation in human primary tumors. The methylation status of these CpG sites was confirmed to be representative of their surrounding CpG islands in human primary tumors and control cerebellar samples, using bisulfite sequencing approaches. We next assessed whether the DNA methylation status of orthologous regions was altered in tumors from four independent mouse models driven by Shh pathway activation (Ptc1+/- Ink4c-/-; Smo/Smo; Sufu+/- p53-/-; p53-/- Ink4c-/-), compared to strain-matched control mouse cerebella. Tissue-specific methylation patterns in the normal cerebellum were conserved between mouse and human, for 10/14 of the candidate regions investigated. Three regions which showed hypermethylation in human medulloblastomas (DSC2, RASSF1A and TAL1) also showed evidence of hypermethylation in mouse tumors and five regions showed evidence of hypomethylation in both human and mouse tumors (CYP2E1, MMP9, SPDEF, TGFB1, and VAV1). CYP2E1 and VAV1 showed the most similar frequencies and patterns of methylation changes in mouse and human tumors, while other changes were either less frequent (RASSF1A), affected fewer CpG sites (TAL1, TGFB1, SPDEF) or had different tissue-specific methylation patterns (DSC2, MMP9) in mice. Ongoing work investigates the relationship of these methylation changes to gene expression and tumorigenesis. Assessment of the conservation of epigenetic changes between human tumors and disease relevant mouse models may help highlight those alterations most important in driving tumorigenesis, and identify candidate genes to take forward for further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3449. doi:10.1158/1538-7445.AM2011-3449