Cell-based Surface Plasmon Resonance Imaging Technique And Its Application On Pharmaceutical Analysis

Surface plasmon resonance(SPR) is being widely employed to determine the binding affinity of drug candidate with its target in chemical and pharmaceutical industry, due to it is of the properties of real-time, label-free and high sensitivity. However, traditional SPR application involves extraction, purification and immobilization of target protein on the sensor chip, which is labor-intensive and problematic, especially for the protein molecules whose conformations are sensitive to the local membrane environment in an intact cell. Thus, we developed the cell-based surface plasmon resonance image(SPRi) technique to directly monitor the binding process of biological molecules or nanoparticles with intact cells.At first, we applied the cell-based SPRi technique to clarify how the fluorescent labeling affects the binding kinetics of proteins with cells, aiming to underscore the importance of label-free method on biochemistry investigation. In this research, we labeled the molecule of wheat germ agglutinin(WGA) with fluorescent tags of different charge properties, and obtained the binding kinetics of fluorescent-labeled and un-labeled molecules(probe molecule) with glycoproteins(target molecule) on the membrane of cells. The comparison among their binding behaviors shows that fluorescent labeling has a significant influence on the association processes of protein molecules and also the distribution of their binding localization. To examine the origin of the effect, we further seperatively obtained the binding kinetics of WGA labeled with positive and nagetive tags in phosphate buffered saline(PBS) solution with different ionic strengths. The results reveal that the effect arises from the charge of the fluorescent labels, which enhances or weakens the binding due to electrostatic interaction between the fluorescent labeled probe molecule and the target molecule on the negatively charged membrane surface. Moreover, due to the inhomogeneous surface charge distribution of the cell membrane, the labels also affect the local distribution of probe proteins.And then, we improved the signal to noise ratio of the experiment setup through stabilizing the temperature of light source and fixing the position of sensor chip. After the improvement, we demonstrated the cell-based SPRi technique could quantify the binding kinetics of drug-target interactions. We measured the binding process of monoclonal antibody drug, Herceptin, with its target molecules, human epidermal growth factor receptor 2(Her2) on different cells, including SKBR3, BT474, MCF7-Her2 and primary cells. The results reveal that the binding kinetics constants of Herceptin-Her2 interaction are inhomogeneous on different cells, especially on the primary cells, which suggesting the results of label-free method on single cells are more accurate and liable. Besides, the detecting of biomarker Her2 on primary cells of early-stage tumor tissue also implies the capacity of this method on disease diagnosis.The Her2-positive cancer patient usually develop drug resistant in the late-stage of Herceptin treatment, thus, next, we measured the in situ Herceptin-Her2 binding kinetics in single intact Herceptin-sensitive and Herceptin-resistance cancer cells. Through comparison between their binding kinetics, we observed significantly weaker Herceptin-Her2 interactions in Herceptin-resistant cells than those in Herceptin-sensitive cells. In conjunction with dual immunofluorescence staining, we further showed that the steric hindrance of Mucin-4, a membrane protein, was responsible for the altered drug-receptor binding.At last, we attempted the SPRi technique on determining the binding affinity of antibody-conjugated nano-medicine with antigen-expressing cells, and compare the results with that of the nanoparticle-free antibody. We find that the nano-conjugates can significantly affect the binding kinetics compared with free antibody molecules, depending on the density of the antibody conjugated on the nanoparticles, and expressing level of the antigen on the cell membrane. The results are analyzed in terms of a transition from mono-valent binding model to a bi-valent binding model when the conjugation density and expressing level increase. And the results also reveal that the nano-conjugates with bi-valent binding could effectively stay on the cell surface and take reaction there.In conclusion, the work in this thesis demonstrats and extends the application of cell-based SPRi technique on biochemical analysis and pharmaceutical engineering. In addition to underscore the importance of label-free investigation on individual cells for biochemical field, we also clarified the effect of molecule charge on the protein interaction for fluorescent labeling method. Besides, we elucidated the molecular mechanism of drug resistance on Her2-posivtive cancer cells at the late-stage of Herceptin treatment through this method, and determined the key factors to improve the binding affinity of nano-medicine on cell surface. These findings not only are beneficial for our understanding on some biochemical process, but also provide some useful information for drug discovery and disease therapy.