Background

[PubMed]

Chlorotoxin (CTX) is a neurotoxin comprising 36 amino acids and is isolated from the venom of Leiurus quinquestriatus, a scorpion of the Buthidae family. A characteristic feature of the peptide is that it contains four disulfide bonds that give it a tight tertiary structure and a single tyrosine residue that can be iodinated. Originally, CTX was described as a calcium channel blocker (1) that was later shown to bind specifically to isoform 2 of a matrix metalloproteinase (MMP2) associated with the channels (2). The MMP2 was shown to be upregulated in many cancers, especially glioblastomas, and there appears to be a correlation between the level of MMP2 expression and poor outcome of the disease (1-6). For most neoplastic conditions, a surgical resection of affected tissue is the most common treatment, and the precision of this technique can influence patient prognosis. The identification and removal of tumors is largely based on a surgical judgment and can be imprecise because it involves avoiding the removal of normal or apparently healthy tissue. Incomplete removal of cancer cells is a problem often encountered with brain tumors, and >80% of malignant cancers recur at the site of tumor removal (7). Clear distinction of tumor tissue from normal tissue could alleviate the problem of incomplete removal of cancer tissue by surgical resection and help improve patient prognosis. The development of agents that bind to tumor-specific molecules and their use to illuminate tumor cells during surgery would greatly facilitate the achievement of this goal.

Biocompatible fluorescent contrast agents conjugated to peptides, proteins, or antibodies, including those visible with near infrared (NIR) light, have been developed and evaluated for the optical imaging of tumors (8, 9). These agents can be used for non-invasive or intraoperative imaging because water or hemoglobin do not absorb in the NIR spectrum and allow the photons to penetrate the tissue. The use of a cyanine (Cy) dye conjugated to a variety of molecules has been explored to image various cancers (10). From these studies it was concluded that small peptides offer a greater advantage over large molecules, e.g., antibodies, because they are easy to synthesize and modify, are likely to be less immunogenic, can be rapidly cleared from circulation, and rapidly accumulate in the target area to give a high signal/background ratio.

Brain tumor imaging has been performed with NIR probes that targeted tumor microglia or required proteolytic activation (11, 12). However, the former approach does not always correlate with detection of the brain tumor margins because patients are often treated with dexamethasone, an inhibitor of microglia activation, to reduce post-operative edema. Previously, a multifunctional probe fabricated with iron oxide nanoparticles coated with covalently bound bifunctional polyethylene glycol, CTX, and Cy5.5 was developed and successfully used invitro to image glioma cells (13). CTX and Cy5.5 are available commercially in the United States. Fluorescence microscopy and magnetic resonance imaging showed that conjugated nanoparticles were taken up preferentially by the glioma cells. From observations made during the study, the potential use of the conjugate for real-time imaging of glioma brain tumors was suggested. Subsequently, Veiseh et al. developed and evaluated a CTX:Cy5.5 dye bioconjugate that requires no protease activation to image neoplastic tumors in a mouse model (14).

Synthesis

[PubMed]

The synthesis of CTX:Cy5.5 was described by Veiseh et al. (14). Both CTX and the Cy5.5 dye (as an ester) were purchased from commercial sources. The Cy5.5 dye was dissolved in anhydrous dimethyl formamide and mixed with a CTX solution in bicarbonate buffer (pH 8.5) at a ratio of 3:1. The conjugation was allowed to proceed for 1 h at room temperature in the dark. Excess dye was removed by dialysis against phosphate-buffered saline (PBS) for 18 h at 4°C. The samples were diluted in PBS to various appropriate dilutions and filter-sterilized for use in the different studies. Quality of the conjugated product was monitored by mass spectroscopy. The yields, purity, and Cy5.5/CTX ratio of the conjugated product were not provided in the publication.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

For in vitro evaluation of CTX:Cy5.5, 9L glioma cells and primary fibroblasts (as controls) were exposed to the same concentration of the bioconjugated agent under identical conditions and the binding was visualized by NIR fluorescence (NIRF) microscopy (14). All the 9L glioma cells exposed to the probe emitted a signal in the NIRF spectrum, and a minimal signal was detected with the fibroblasts. It was also observed that the MMP2 inhibitor 1,10-phenanthroline blocked the binding of CTX:Cy5.5 to the 9L glioma cells, which indicated that the CTX:Cy5.5 bioconjugate bound specifically to the metalloproteinase.

In another study to show that MMP2 is specifically involved in the binding of CTX:Cy5.5, MCF7 cells known to express low levels of MMP2 and bond CTX:Cy5.5 poorly compared to the other cell lines were transfected with a plasmid encoding the MMP2 (14). Cells expressing the MMP2 were sorted from the untransfected cells and an increased expression of the proteinase in these cells was confirmed by indirect immunofluorescence, Western blot analysis, and gelatinase assays. Although no signal/noise ratios were provided by the investigators, from image analysis it was clear the MMP2-transfected cells showed higher CTX:Cy5.5 binding compared to control cells transfected with the control plasmid (which contained no MMP2 insert). This again confirmed that CTX:Cy5.5 bound specifically to MMP2.

Animal Studies

Human Studies

[PubMed]

No publications are currently available.

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