Microelectrode-/microscopic-image-based Method For Cancer Therapeutic Drug Screening

High throughput screening (HTS) is the critical technology in modern drug discovery. It is widely used in discovering hit from the chemical library in primary screening, and in analyzing the selectivity and toxicity of lead. It also plays an important role in hit validation and lead optimization.Cancer multidrug resistance (MDR) is a major impediment to effective chemotherapy in human cancer. A large body of research spanning some two decades in search of direct MDR inhibitors has so far failed to provide an agent effective in clinical application. Design and, exploitation of novel clinical MDR inhibitors is greatly hindered by a lack of understanding of drug efflux dynamics in drug-sensitive and resistant cells. Because the micro-cell-sensor takes advantage of the high sensitivity and resolution of the micro-analysis, it is more suitable for determining real-time anticancer drug efflux from intact cell population. So we propose a novel carbon-fiber-microelectrode-based method for monitoring doxorubicin (Dox) efflux from monolayer cancer cells and screening MDR reversal agent.High content screening (HCS) is an image-based method to acquire physical or pathological properties of cells from cellular image. Unlike the measurements averaged over the well used in traditional HTS, HCS is based on investigation of the cellular image, so it is more applicable for studying the sub cell population or sub-cell structure. In the present work, we provide an exploratory work in developing a digital image microscopy system for automatic high content screen (Di-HCS).The major contents and contributions of this thesis are given in the following aspects:(1) Our study provided a carbon-fiber-microelectrode-based method to measure local instantaneous concentrations of the efflux of Dox molecules from monolayer MCF-7 and MCF-7/ADR cells.(2) We first applied the inverse method to a general model for the biomaterial diffusion process from the monolayer cells and obtained insights regarding kinetic parameters through the quantitative estimation. A computer program was written for numerical simulation of the corresponding mathematical model that allowed calculation of mass transport as a function of time and position. We were able to quantitatively compare the efficacy of several potential MDR reversal agents and understand the kinetics of drug efflux from drug-sensitive and resistant cancer cells. Our results suggest more opportunities for the computer-aided reconstruction of the drug efflux process for quantitative pharmacokinetic effects of MDR inhibitors on anticancer drugs.(3) We also completed the construction of hardware and the primary software development of Di-HCS. Using Di-HCS and different fluorescent dyes, three microscopic-image-based drug screening assays are proposed: FDA based cell viability assay; Hoechst 33342 and propidium iodide (PI) based apoptosis assay; FDA, Hoechst 33342 and PI based apoptotic pattern recognition assay.