Tsourkas, Andrew
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Publication Superparamagnetic Iron Oxide Nanoparticle Probes for Molecular Imaging(2006-01-01) Thorek, Daniel L. J; Chen, Antony K; Czupryna, Julie; Tsourkas, AndrewThe field of molecular imaging has recently seen rapid advances in the development of novel contrast agents and the implementation of insightful approaches to monitor biological processes non-invasively. In particular, superparamagnetic iron oxide nanoparticles (SPIO) have demonstrated their utility as an important tool for enhancing magnetic resonance contrast, allowing researchers to monitor not only anatomical changes, but physiological and molecular changes as well. Applications have ranged from detecting inflammatory diseases via the accumulation of non-targeted SPIO in infiltrating macrophages to the specific identification of cell surface markers expressed on tumors. In this article, we attempt to illustrate the broad utility of SPIO in molecular imaging, including some of the recent developments, such as the transformation of SPIO into an activatable probe termed the magnetic relaxation switch.Publication Sub-cellular trafficking and functionality of 2'-O-methyl and 2'-O-methyl-phosphorothioate molecular beacons(2009-10-09) Chen, Anthony K; Tsourkas, Andrew; Behlke, Mark AMolecular beacons (MBs) have shown great potential for the imaging of RNAs within single living cells; however, the ability to perform accurate measurements of RNA expression can be hampered by false-positives resulting from nonspecific interactions and/or nuclease degradation. These false-positives could potentially be avoided by introducing chemically modified oligonucleotides into the MB design. In this study, fluorescence microscopy experiments were performed to elucidate the subcellular trafficking, false-positive signal generation, and functionality of 2'-O-methyl (2Me) and 2'-O-methyl-phosphorothioate (2MePS) MBs. The 2Me MBs exhibited rapid nuclear sequestration and a gradual increase in fluorescence over time, with nearly 50% of the MBs being opened nonspecifically within 24 h. In contrast, the 2MePS MBs elicited an instantaneous increase in false-positives, corresponding to ~5–10% of the MBs being open, but little increase was observed over the next 24 h. Moreover, trafficking to the nucleus was slower. After 24 h, both MBs were localized in the nucleus and lysosomal compartments, but only the 2MePS MBs were still functional. When the MBs were retained in the cytoplasm, via conjugation to NeutrAvidin, a significant reduction in false-positives and improvement in functionality was observed. Overall, these results have significant implications for the design and applications of MBs for intracellular RNA measurement.Publication Suicide Gene Delivery by Calcium Phosphate Nanoparticles: A Novel Method of Targeted Therapy for Gastric Cancer(2006-12-01) Tsourkas, Andrew; Czupryna, JuliePublication Size, charge and concentration dependent uptake of iron oxide particles by non-phagocytic cells(2008-05-27) Thorek, Daniel L.J.; Tsourkas, AndrewA promising new direction for contrast-enhanced magnetic resonance (MR) imaging involves tracking the migration and biodistribution of superparamagnetic iron oxide (SPIO)-labeled cells in vivo. Despite the large number of cell labeling studies that have been performed with SPIO particles of differing size and surface charge, it remains unclear which SPIO configuration provides optimal contrast in non-phagocytic cells. This is largely because contradictory findings have stemmed from the variability and imprecise control over surface charge, the general need and complexity of transfection and/or targeting agents, and the limited number of particle configurations examined in any given study. In the present study, we systematically evaluated the cellular uptake of SPIO in non-phagocytic T cells over a continuum of particle sizes ranging from 33 nm to nearly 1.5 μm, with precisely controlled surface properties, and without the need for transfection agents. SPIO labeling of T cells was analyzed by flow cytometry and contrast enhancement was determined by relaxometry. SPIO uptake was dose-dependent and exhibited sigmoidal charge dependence, which was shown to saturate at different levels of functionalization. Efficient labeling of cells was observed for particles up to 300 nm, however, micron-sized particle uptake was limited. Our results show that an unconventional highly cationic particle configuration at 107 nm maximized MR contrast of T cells, outperforming the widely utilized USPIO (<50 nm).Publication Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells(2007-08-15) Chen, Antony K; Behlke, Mark A; Tsourkas, AndrewThere have been a growing number of studies where molecular beacons (MBs) are used to image RNA expression in living cells; however, the ability to make accurate measurements can be hampered by the generation of false-positive signals resulting from non-specific interactions and/or nuclease degradation. In the present study, we found that such non-specific signals only arise in the nucleus of living cells. When MBs are retained in the cytoplasmic compartment, by linking them to quantum dots (QDs), false-positive signals are reduced to marginal levels. Consequently, MB–QD conjugates were used to measure the expression of the endogenous proto-oncogene c-myc in MCF-7 breast cancer cells by quantifying the total fluorescent signal emanating from individual cells. Upon the addition of tamoxifen, measurements of MB fluorescence indicated a 71% reduction in c-myc expression, which correlated well with RT-PCR measurements. Variations in MB fluorescence resulting from instrumental fluctuations were accounted for by imaging fluorescent calibration standards on a daily basis. Further, it was established that measurements of the total fluorescent signal were not sensitive to the focal plane. Overall, these results provide evidence that accurate measurements of RNA levels can be made when MBs are retained in the cytoplasm.Publication Efficient cytosolic delivery of molecular beacon conjugates and flow cytometric analysis of target RNA(2008-05-08) Chen, Antony K; Behlke, Mark A; Tsourkas, AndrewFluorescent microscopy experiments show that when 2’-O-methyl-modified molecular beacons (MBs) are introduced into NIH/3T3 cells, they elicit a nonspecific signal in the nucleus. This false-positive signal can be avoided by conjugating MBs to macromolecules (e.g. NeutrAvidin) that prevent nuclear sequestration, but the presence of a macromolecule makes efficient cytosolic delivery of these probes challenging. In this study, we explored various methods including TAT peptide, Streptolysin O and microporation for delivering NeutrAvidin-conjugates into the cytosol of living cells. Surprisingly, all of these strategies led to entrapment of the conjugates within lysosomes within 24 h. When the conjugates were pegylated, to help prevent intracellular recognition, only microporation led to a uniform cytosolic distribution. Microporation also yielded a transfection efficiency of 93% and an average viability of 86%. When cells microporated with MB–NeutrAvidin conjugates were examined via flow cytometry, the signal-to-background was found to be more than 3 times higher and the sensitivity nearly five times higher than unconjugated MBs. Overall, the present study introduces an improved methodology for the high-throughput detection of RNA at the single cell level.Publication Radiometric Bimolecular Beacons for Sensitive Detection of RNA in Single Living Cells(2010-05-27) Davydenko, Olga; Chen, Antony K; Tsourkas, Andrew; Behlke, Mark ANumerous studies have utilized molecular beacons (MBs) to image RNA expression in living cells; however, there is growing evidence that the sensitivity of RNA detection is significantly hampered by their propensity to emit false-positive signals. To overcome these limitations, we have developed a new RNA imaging probe called ratiometric bimolecular beacon (RBMB), which combines functional elements of both conventional MBs and siRNA. Analogous to MBs, RBMBs elicit a fluorescent reporter signal upon hybridization to complementary RNA. In addition, an siRNA-like doublestranded domain is used to facilitate nuclear export. Accordingly, live-cell fluorescent imaging showed that RBMBs are localized predominantly in the cytoplasm, whereas MBs are sequestered into the nucleus. The retention of RBMBs within the cytoplasmic compartment led to >15-fold reduction in false-positive signals and a significantly higher signal-to-background compared with MBs. The RBMBs were also designed to possess an optically distinct reference fluorophore that remains unquenched regardless of probe confirmation. This reference dye not only provided a means to track RBMB localization, but also allowed single cell measurements of RBMB fluorescence to be corrected for variations in probe delivery. Combined, these attributes enabled RBMBs to exhibit an improved sensitivity for RNA detection in living cells.