Targeting Anti-gliomas And Tumor Diagnosis Of The Recombinant Scorpion Chloride Channel Toxin

Gliomas, which are a primary tumor originated in the glial cells, are the most common and difficult to treat malignant tumor in the central nervous system. They have high deadth rate and the average survival time of patients are less than a year. Even more seriously, the incidence of malignant glioma continuously rises in recent years, becoming the second cause of death below34years old patients. It is difficult to diagnosis gliomas by the traditional iconography, because of their invasive growth and the unobvious boundary in the early stage, and rapid proliferation and high level of invasion and metastasis of malignant glioma, resulting in the lack of effective methods in diagnosis and treatment, In modern medical studies, gliomas express a special kind of chlorine ion channel current which is different with other tumor cells. Studies have shown shows that some scorpion toxins (such as chlorotoxin (CTX), etc) can highly specifically bind to the cell membrane metal matrix protease2(MMP-2), and can inhibit glioma proliferation and metastasis by blocking the chlorine ion channel current. With the recent rapid development of nanotechnology, nanoplatform-based fluorescent probe detection technology is taken more and more attention, becoming the focus of the research in the cross field of optical and life sciences.In this paper, the CTX nucleotide sequence was generated from the primary sequence of natural CTX peptide (36amino acid residues) by an overlapping PCR method. The PCR products were cloned into pGEX-6p-1to construct the expression vector pGEX-6p-1-CTX plasmid which was then transformed into E. coli Rosetta (DE3) cells. The expression of the recombinant GST-CTX fusion protein (gCTX) was induced by isopropy-P-D-thiogalactoside (IPTG). The recombinant fusion protein was purified by GSH affinity chromatography column. After the purified fusion protein was desalted using centrifugal filtration and cleaved by enterokinase, the recombinant CTX peptide was purified by high performance liquid chromatography (HPLC). The yield of the recombinant CTX was1.5mg/L culture and its purity was more than95%. The molecular weight of the recombinant CTX peptide was3998.03Da, which was identified by mass spectrometry. The result is consistent with its theoretical molecular weight3997.58Da. We developed a recombinant system to achieve production of the high purified scorpion chloride channel toxin CTX peptide.The recombinant CTX was conjugated with rare-earth upconversion fluorescence nanoparticles after surface amination modification. The scorpion toxins-rare-earth upconversion fluorescence nanoprobe (CTX:UCNPs) was prepared. The physical properties of the probe were systematically tested for the fluorescence intensity, fluorescence stability and cell toxicity. To build a upconversion living imaging system, and finish the targeted labelling and visual detection of subcutaneous xenogenous glioma in mice, systematic study of CTX:UCNPs targeted label the glioma cells in vitro and living tumor in vivo. The results showed that the synthesized probe can realize targeted fluorescence imaging and visual detect of living tumors when exposed in the near infrared.We also used the same recombinant expressing method to obtain highly pure scorpion chloride channel toxin gCTX fusion protein. A glioma-bearing model of rat was established to investigate targeting inhibition experiment of the proliferation and metastasis of gCTX protein against glioma. The data showed that the volume and weight of the gCTX-treated tumors were significantly less than those of the control group. The growth inhibition rate of glioma was85.45±3.41%after a17-days treatment of gCTX, while the metastasis inhibition rate of glioma with gCTX protein reached50%after27-days treatment. Through labelling gCTX protein with131I or Cy5.5, we found that the recombinant fusion protein gCTX can specifically target glioma in vivo.Based on the tagertd anti-glioma activity of gCTX protein, it was used as a template to synthesize the glioma targeted and visible near infrared fluorescent toxin-gold nanoclusters compounds. The physical properties of the probe were systematically tested for the fluorescence intensity, fluorescence stability and cell toxicity. And then, the function of targeted inhibition of the proliferation and metastasis against gliomas was studied. The results showed that the probe Au@(gCTX) NCs can target glioma cells, and inhibit growth and MMP-2activity of glioma cells in vitro, whose inhibitory rates were42.23±1.51%and60.34±0.05%, respectively. Moreover, this probe also inhibited the invasion and metastasis of gliomas.Combined with the highly specific glioma-targeting of scorpion chlorine toxin and unique biological fluorescence property of nanoparticles, two scorpion toxin fluorescence nanoprobes were designed and developed, possibly offering a kind of effective new tools for the pathogenesis, molecular imaging diagnosis and clinical treatment of tumor. They may have a potential value in bioapplication.