There is currently interest in the development of nanoemulsions as imaging and therapeutic agents, particularly perfluorohexane (PFH) droplets, whose amphiphilic shell protects drugs against physico-chemical and enzymatic degradation. When delivered to their target sites, these perfluorocarbon (PFC) droplets can vaporize upon laser excitation, efficiently releasing their drug payload and/or imaging tracers. Due to the optical properties of gold, coupling PFC droplets with gold nanoparticles significantly reduces the energy required for vaporization. In this work, nanoemulsions with a PFC core and Zonyl FSP surfactant shell were produced using sonication. Droplets were characterized in terms of size and morphology using high resolution fluorescence microscopy (i.e. total internal reflection fluorescence microscopy, TIRFM), fluorescence correlation spectroscopy (FCS), transmission electron microscopy (TEM), and light scattering techniques (i.e. dynamic light scattering, DLS). The ability of PFC droplets to vaporize was demonstrated using optical light microscopy.
Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein plays an important role in the onset of cancers such as leukemia and lymphoma. In this study, we aim to test the effectiveness of a novel peptide drug designed to tether STAT3 to the phospholipid bilayer of the cell membrane and thus inhibit unwanted transcription. As a first step, STAT3 proteins were successfully labelled with tetramethylrhodamine (TMR), a fluorescent dye with suitable photostability for single molecule studies. The effectiveness of labelling was determined using fluorescence correlation spectroscopy in a custom built confocal microscope, from which diffusion times and hydrodynamic radii of individual proteins were determined. A newly developed fluorescein derivative label (F-NAc) has been designed to be incorporated into the structure of the peptide drug so that peptide-STAT3 interactions can be examined. This dye is spectrally characterized and is found to be well suited for its application to this project, as well as other single-molecule studies. The membrane localization via high-affinity cholesterol-bound small-molecule binding agents can be demonstrated by encapsulating TMR-labeled STAT3 and inhibitors within a vesicle model cell system. To this end, unilaminar lipid vesicles were examined for size and encapsulation ability. Preliminary results of the efficiency and stability of the STAT3 anchoring in lipid membranes obtained via quantitative confocal imaging and single-molecule spectroscopy using a custom-built multiparameter fluorescence microscope are reported here.
The optical properties and significant surface area of CdSe/ZnS QDs make such nanoparticles an interesting platform for
the preparation of nucleic acid biosensors based on fluorescence resonance energy transfer (FRET). Interactions between
QDs and oligonucleotides affect biosensor performance and are not fully understood. Ensemble data obtained via FRET
experiments indicated that, on average, 4-5 added oligonucleotides saturated the surface of green emitting QDs. An
increase in the number of oligonucleotides per QD appeared to cause the oligonucleotides to transition from collapsed to
upright conformations. Since bulk averaging hides details of such processes, methods must be developed and materials
identified for studying QD-oligonucleotide conjugates at the single molecule level. Single QDs have been immobilized
and fluorescence intensity trajectories measured. High count rates and good photostability were achieved using carboxyl
polymer-coated QDs. Modeling of FRET efficiency based on the dimensions of QDs and oligonucleotides indicated that
transitions between collapsed and upright conformations can be accurately measured based on changes in QD
fluorescence lifetime. The ultimate goal of this work is to elucidate QD-oligonucleotide dynamics for better design and
optimization of nucleic acid biosensors based on QDs.