Nance, ElizabethZhang, Mengying2021-03-192021-03-192021-03-192020Zhang_washington_0250E_22299.pdfhttp://hdl.handle.net/1773/46716Thesis (Ph.D.)--University of Washington, 2020Central nervous system (CNS) diseases have long known hard to treat due to (1) highly restricted barriers such as the blood brain barrier (BBB) and the brain parenchyma penetration barrier, and (2) the dynamic and heterogeneous neurological disease microenvironment, which is dependent on disease etiology and disease progression, and is variable from patient to patient. Nanotechnology based imaging probes, such as quantum dots (QDs), are thus of interest to study complex changes in the brain and develop cutting-edge imaging tools. QDs have several advantages over traditional probes including tailorable excitation and emission spectra, tailorable surface functionalities, and high photoluminescence and photostability, which are ideal characteristics for in vivo imaging. QDs have been utilized in various applications in the brain; however, a systematic evaluation of QD behavior in brain-relevant conditions has not been done. Therefore, we first sought to comprehensively investigate QD colloidal stability, toxicity, and cellular uptake in vitro, ex vivo and in vivo in the neonatal brain environment. We found QD behavior is highly dependent on surface functionality, and QDs can activate metallothionein detoxification pathways in cells in organotypic whole hemisphere slice cultures. QDs are mainly internalized in the corpus callosum region in microglia, cells that mediate inflammation in the brain. Using multiple biological models in this study also indicated the need for careful consideration of barriers that exist in different study platforms, when investigating nanomedicine behavior in brain microenvironment.We next sought to utilize QDs to label and visualize extracellular vesicles (EVs). EVs are cell-secreted vesicles that play an important cell-cell communication role and participate in normal and pathological processes. Direct visualization and tracking of EVs is essential to broaden the utility of EVs as biomarkers of injury, however, current labeling techniques fail due to lack of sensitive, specific and robust labeling. We have developed a QD-EV conjugation platform using 4-formylbenzoate (4FB) to 6-hydrazinonicotinate acetone hydrazone (HyNic) click chemistry. We showed this method can be performed under physiological conditions, is universal to EVs coming from different sources, such as human semen fluids and rat brain, and has better resistance to photobleaching compared to commonly used labeling dye. This QD-EV conjugation method also has room for optimization by using catalyst or altering QD:EV ratio. QD-EV conjugates and unconjugated QDs can be easily separated by size exclusion chromatography (SEC). We also show this QD-EV conjugation platform enables high resolution visualization and real-time study of the EV-cell interaction. In summary, we investigated both fundamental understanding of QD neuroimaging probes in the developing brain and application of QDs for labeling and visualizing EVs. The understanding of QD behavior and impact in the brain, and subsequent use of QDs to label and trace biological entities represents a promising advancement in the field of bioimaging for neurological disease.application/pdfen-USnonecentral nervous systemclick chemistryextracellular vesiclesmicroglianeuroimagingquantum dotsNanotechnologyBiomedical engineeringNeurosciencesMolecular engineeringThesis