Evaluation Of Tumor Resistance Based On Cell Impedance Sensing Technique

Lung cancer is the most common malignant tumor, compared with surgery or radiotherapy,chemotherapy has an irreplaceable status as a tool of systemic treatment for malignant tumor. The cell-based in-vitro drug screening methods are effective measures for preclinical screening of antitumor drugs. MTT assay is the most commonly used screening method. However MTT has certain limitations, it is an endpoint test which can’t keep a continuous and real-time monitoring. By measuring the electrical impedance changes between the cells and microelectrodes in a simulated physiological environment Electrical, cell-substrate impedance sensing(ECIS) can be used to record the cell proliferation, cytotoxicity and changes in cell morphology dynamically. It has the advantages such as label-free, noninvasive, real-time monitoring, high sensitivity, and high throughput. The purpose of this scientific research is to establish a new method for in-vitro drug screening and evaluation with ECIS, and apply it to the evaluation of Silybin-phospholipid soluble particulates which reverse resistance of A549 / PTX cell.In this research, the lung adenocarcinoma cell A549 and human large-cell lung cancer cell NCI-H460 was used as the experimental cell. First of all, the linear relationship between inoculation amount and the absorbance were measured to evaluate the efficiency of the MTT method. Then the sensitivity of cell A549 and cell NCI-H460 under the effect of four antitumor drugs including paclitaxel, docetaxel, vinorelbine pemetrexed and cis-platinum were measured. Both cells were sensitive to paclitaxel and docetaxel. Cell inhibition rate was increased with the increase of drug concentration. Cell A549 was poor sensitive to vinorelbine and pemetrexed. Conversely, the sensitivity of NCI- H460 cell to the two drugs were moderate, But with the increase of drug concentration the inhibition rate of the both cell had not changed obviously. Under the operation of cisplatin, inhibition rate of the A549 cell increased with the increase of drug concentration. However the inhibition rate of NCI-H460 cell had not changed obviously with the increase of drug concentration.ECIS and MTT assay were respectively used to determine the effects of four antitumor drugs including paclitaxel, docetaxel, vinorelbine and pemetrexed on the growth of human large-cell lung cancer cell line NCI-H460. The data analysis methods such as paired t-test, intraclass correlation coefficient and Bland-Altman method were utilized to comprehensively evaluate the consistency of the inhibition rates measured by the two methods. Cell morphology observations under electron microscope and verification test with the human lung adenocarcinoma cell line A549 were combined to assess the new method. Results were obtained as followed. The dynamic information for the effects of various drugs on cell line NCI-H460 and A549 can be characterized qualitatively and quantitatively by ECIS. The dynamic characteristic information of the inhibitory effects of various drugs on NCI-H460 cells can be sensitively reflected by the dynamic biological response spectrum characterized by the cell index-response time correlation. Furthermore, the analysis results of inhibition rates showed that the two methods were consistent. The morphologies of NCI-H460 cells in various phases under electron microscope and the verification tests with A549 cells further indicated the feasibility of the method. So the ECIS-based method can be used for in-vitro drug screening and evaluation.Lastly, Silybin-phospholipid soluble particulates were formulated with Silymarin, lecithin, and other excipients and characterized by appearance, solubility and content. And its cytotoxicity was determined using the ECIS method to screen a drug dose under which the inhibition rate for A549/PTX cell was less than 20%. The drug dose applied to drug resistance study. At the same time, the dynamic biological response spectrum and inhibition rate of A549 cell and A549/PTX cell under the effect of paclitaxel can be detected by the ECIS-based method. The sensitivity of A549 / PTX cell under the effect of paclitaxel is significantly lower than that of A549 cell,but showed a strong resistance. When drug paclitaxel was given with a certain concentration, the similarity was showed to the biological response spectrum and showed a peak valley trend in this two cell lines. The peak time of the dynamic biological response spectrum forwarded about 5h and the inhibition rate increased to a certain degree. It was concluded that Silybin-phospholipid soluble particulates could reverse resistance of A549 / PTX cell.