Development of fluorescent semi-conductor nanocrystal conjugates for in vitro and in vivo imaging applications

Abstract

Semiconductor nanocrystals, also known as quantum dots (QDs), are promising imaging probes with characteristic optical properties: tunable bandgap from visible to infrared, narrow and symmetric emission features, broad absorption, high quantum yield (QY), excellent photostability, and a large two-photon absorption cross section. However, unlike other imaging probes, the surface ligands determine the solubility, stability, quantum yield (QY), biocompatibility, and derivatizability of QDs. Therefore, to use QDs for in vitro and in vivo imaging, QD ligands need to be elaborately engineered for each experiment. Single cell labeling in vivo requires extremely strict criteria for the QD conjugates to be used such as minimal nonspecific cell/serum binding, maintenance of high QY in complex in vivo environments, and compact size. The focus of this thesis is the synthesis of high quality QD conjugates that can be used for single molecule imaging in vivo and in vivo imaging studies that demonstrate the broad and powerful applicability of our new methods. We incorporated novel conjugation methods employing highly strained cycloolefins and a serum stable tetrazine derivative into newly developed polymeric imidazole ligands (PILs) to efficiently couple biomolecules on QDs. Unlike traditional conjugation methods, tetrazine-norbornene cycloaddition benefits from the non-interacting properties of the functional groups to the QD surface, and yields the high conjugation efficiencies on QDs. In addition, the rapid kinetics, absence of catalyst, and bio-orthogonality of the cycloaddition allowed us to achieve in situ conjugation of the norbornene-bearing QDs to tetrazine-bearing epidermal growth factor (EGF) proteins on the HeLa cell surface. On the in vivo front, we accomplished single endogenous cell imaging in live mice. The ability to target single cells using multiple biomarkers and track them for the extended periods of time allowed us to study the microenvironment of the endogenous hematopoietic stem cells (HSCs), which was not possible using conventional techniques engaging dye conjugated antibodies. Lastly, a new class of QD ligands containing betaine moieties was developed to reduce the size of QD conjugates, which we expect will be greatly beneficial for in vitro and in vivo targeting in dense environments. We successfully demonstrated functionalization of the sulfonate betaine poly imidazole ligands (SBPILs) with biomolecules, and the biocompatibility of SBPIL QDs both in vitro and in vivo.