Development of Nanoparticle-Based Intracellular Dual Sensing and Actuation Modalities

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The integration of therapeutics with diagnostic agents, or theranostics, is vital for the development of novel and effective disease treatments. To effectively design new and efficient theranostic materials, a thorough understanding of the carrier ensemble, the interactions within the construct components, and the surrounding environment is required. This dissertation focuses on the development of new strategies to produce an effective ‘toolbox’ of nanoscale theranostics, namely through the use of a central NP scaffold and the visualization technique of Förster Resonance Energy Transfer (FRET). The NP scaffold used throughout this work, the semiconductor quantum dot (QD), is ideal for visualizing sensing modalities due to their high quantum yield (QY), tunable, narrow and symmetric emission profiles with broad, far-UV excitation, and resistance to photobleaching - making them optimal FRET donors. We first examined the intracellular assembly of QDs to proteins by injecting 545 nm emitting QDs, coated with various capping ligands, into cells transfected to express mCherry at two distinct intracellular locations: the cytosol and the plasma membrane. We found that the small, zwitterionic capping ligand CL4 and the cytosolically located mCherry protein assembled into the most efficient FRET complexes. We used this knowledge to design and implement a novel intracellular actuation modality for drug delivery that used a 520 nm emitting QD with the carrier maltose binding protein appended to the surface and carrying drug or dye conjugated to a maltose analog, -cyclodextrin in the binding pocket. Rather than relying on intracellular environmental changes or external stimuli to actuate release, the addition of the innocuous sugar maltose to the medium induced cargo actuation and could be visualized via FRET. Finally, the same methods were implemented to develop a novel pH sensor to report on the extracellular changes that occur in tumor development where the physiological pH is lowered dramatically. Using a 464 nm QD scaffold conjugated to pH-responsive FITC, we successfully monitored changes in extracellular pH and accurately determined unknown pH values. With the work in this thesis, we believe we have contributed greatly to the advancement and development NPs for the design and implementation of sensing and actuation complexes.