Tracking Anisotropic Optical Tracers to Study Biophysical Processes and Cytotoxicity

We use anisotropic optical tracers (also called magnetically modulated optical nanoprobes – MagMOONs or MOONs for non-magnetic nanoprobes in this dissertation) to study biophysical processes such as enzyme-catalyzed cleavage through tissue, intracellular transport of these tracers and cytotoxicity based on this transport. The anisotropic optical properties cause these tracers to blink when rotating. This blinking is distinguishable from the background and can be tracked on a single-particle level in the absence of tissue, or for an ensemble average of tracers blinking through tissue. An alginate gel containing these tracers in the form of a thin film can be used as a sensor to detect alginate lyase, a protease of alginate gel. As the protease cleaves the gel, the tracers are released, free to rotate and give a blinking signal that can be tracked under the microscope. The tracers started blinking approximately 10 minutes after 2 mg/mL alginate lyase addition, and this blinking was clearly detected through up to 4 mm of chicken breast. Similar tracer-integrated gel films may potentially be employed to detect bacterial biofilm formation on medical implants by sensing specific proteases that either activate a related function or regulate biofilm formation. It can also be applied to other biosensors and drug delivery systems based on enzyme-catalyzed breakdown of gel components. For intracellular transport and cytotoxicity, we apply the advantages of rotational and translational single particle tracking in cytotoxicity studies by observing the tracers' behavior with and without the presence of toxic substances. Both cyanide and 2-deoxy-D-glucose or azide and 2-deoxy-D-glucose combinations immediately inhibited iii intracellular motion in J774A.1 macrophages upon addition. This result suggested our method can potentially be applied to study cytotoxicity of particulate matter. More importantly, tracking simultaneously the tracers' translation and rotation reveals interesting information about macrophage intracellular transport; for instance, tracers do not rotate when sliding along microtubules. The data analysis also confirmed that sliding periods contributed to a major portion of total movement but comprised a very small portion of total observation time. iv DEDICATION This work is dedicated to my parents, Mr. Nguyễn Văn Thịnh and Mrs. Lê Thị Đon, who have always supported me and been my source of energy, and my brother Nguyễn Tuấn Anh who first introduced me to the world of Science and Chemistry. v ACKNOWLEDGMENTS I would like to specially thank my advisor-mentor, Dr. Jeffrey Anker, for his continuing guidance and support throughout my time doing research …