Construction Of Magnetic Nano-antisense Probe Using For Tumor Multidrug Resistance

Background:In tumor treatment, the chemotherapy occupies the status of an irreplaceable. Since the chemotherapeutic drugs used in clinical, although a small number of malignant tumor treatment has been successful, but the effect in most malignant tumors is not great, which is a very important reason is that the chemotherapeutic drug resistance of tumor cells. And tumor-resistant is mainly due to the over-expression of resistance gene of tumor cells to produce the relevant proteins, such as: lung resistance protein (LRP), multidrug resistance-associated protein (MRP), P-glycoprotein (P-gp). These three resistance protein are LRP gene, MRP gene and MDR1 gene expression products. MDR1/P-gp mediated tumor multidrug resistance is the most extensive and in-depth study of the mechanism. The most direct reason of tumor multidrug resistance (MDR) is MDR1 gene encoded P-gp, so the detection of MDR1/p-gp in cancer chemotherapy patients is extremely important. At present, the detection methods of P-gp and MDR1 are mainly two types, one way is protein assay, another is mRNA test. However, these detection methods must depends on the tumor tissue sample at first, and not only brought great suffering to patients, but also by the expression of MDR1 in the cell, as well as the sensitivity of detection methods and other factors. From the molecular level how to achieve non-invasive evaluate multi-drug resistance of tumor, for the multi-drug resistance monitoring of clinical oncology will be a great breakthrough.Magnetic iron oxide nanoparticles be provided with superparamagnetic, nano-scale, and can be used as gene vector characteristic. MDR1 antisense oligodeoxynucleotide refers to a section of DNA fragments with complementary base pairs and combination to MDR1 gene. Magnetic iron oxide nanoparticles as gene vectors guide MDR1 antisense oligodeoxynucleotide into somatic cells, the application of magnetic resonance technology to detect the magnetic signal in cells to evaluate MDR1 expression in entities tumor tissue is achieved. From the molecular level to achieve non-invasive evaluate tumor entities of multi-drug resistance is possible.Preliminary studies in this experiment show that the magnetic iron oxide nanoparticles are high purity, consistent particle size, and high rate of cell marker. Human colon cancer cell line (LS174T, HCT-8/VCR) were experiment in this study, the synthetic iron oxide magnetic nanoparticles were used, to build MDR1 antisense oligodeoxynucl- eotide vector and in vitro and in vivo experiments, to study the iron oxide Magnetic nanoparticles as gene vector.Objective:Synthesis, Identification of magnetic iron oxide nanoparticles MDR1 antisense oligodeoxynucleotide complex and test cell transfection in vitro, evaluate the feasible of magnetic iron oxide nanoparticles as gene vector.Methods:1. PAGE evaluated DCIONP and MDR1asODN complex, as well as the stability of this complex under different conditions.2. Flow cytometry determined P-gp expression.3. MRI measured magnetic signal of labeled cells.4. Detection of cell transfection: test stained blue iron particles by iron staining cells, fluorescent microscopic observed intracellular fluorescence signal, transmission electron microscopy detected iron particles of the transfected cells.Results:1. PAGE showed DCIONP with MDR1 asODN at different pH have good binding capacity; at weak alkaline conditions, with a certain mass ratio PLL-CIONP and MDR1 asODN have a good binding capacity; in human serum, respectively, running water, and incubated under the conditions of the performance of 37℃the complex is stabilization.2. Determination of flow cytometry results suggested that the fluorescence intensity of LS174T cells was significantly higher than HCT-8/VCR group. It showed that the P-gp was higher expression in HCT-8/VCR cells than that in LS174T cells.3. MRI determination of the magnetic signal of labeled cells showed that as the cells increase in the number of labeled cells of the T2 value of the magnetic signal gradually weakened.4. Cell transfection showed that the staining blue particles of LS174T cell were less than HCT-8/VCR, the fluorescence intensity of LS174T cell was less than HCT-8/VCR by fluorescence microscope, TEM observation were round particles, uniformly distributed.Conclusions:Magnetic iron oxide nanoparticles with MDR1 antisense oligodeoxynucleotides are stable complex, which can be used as gene vector to transfect MDR1 antisense oligodeoxynucleotides. Magnetic iron oxide nanoparticles with 1.5T magnetic resonance medical instrument can detect signals.