Curriculum Vitae – Robin L. McCarley - LSU Department of ...

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Robin L. McCarley September 2011 enzyme shown to be upregulated in roughly half of the NCI cancer cell line database. Current and Future Work. Our investigations are focused on making the quinone trigger group highly susceptible to reduction by quinone oxidoreductase enzymes but at the same time not responsive to other reductases or chemical reducing species. This is being achieved by synthetically manipulating the structure of the quinone trigger so that control over quinone reduction potential is the result, Table 1. We have found that DTT (E1/2 = 0.33 V) is able to reduce all of the currently available quinone propionic acids Qxy-COOH, while glutathione (E1/2 = 0.22 V) and ascorbate (E1/2 = +0.051 V) reduce only QHMe-COOH and QBrMe-COOH. We have developed a multi-well plate assay and are now examining the human NAD(P)H quinone oxidoreductase type 1 (NQO1) activity of these variously substituted trigger groups and have found that the rate of enzymatic reduction of quinone triggers increases with more negative reduction potential, Table 1. In addition, we are making liposomes from the various quinone head groups and studying their rates of contents release (Figure 2, Top). Furthermore, PEG-PE and fusogenic PE lipids are being blended with the QR1-DOPE liposomes to move us toward in vitro studies with cancer cell lines that overexpress NQO1 (Figure 2, Middle). Finally, we have discovered that the opening of liposomes is exquisitely sensitive to the solution environment, with salting in/salting out (Hofmeister) effects being quite prominent, (Figure 2, Bottom). In total, this work will allow us to tune the nature of contents release for long-term drug release studies wherein multiple drugs can be released in a temporally programmed fashion. To assess enzyme distribution in cancer cell lines and in tumors, we are making stimuli-responsive “cloaked” or latent dyes that become highly fluorescent when acted upon by a target enzyme or chemical agent. This a rapidly growing component of our research program, as the idea is one that can be applied to a variety of diseases and target stimuli, both of which are numerous. For example, we have made a cell membrane-impermeable latent Rhodamine dye, and a similar dye that is taken up endocytotically 2- Stimulus % Maximum of S 2 O 4 100 90 80 70 60 50 40 Dithionite 30 20 DTT GSH AA 10 0 + PBS 0 50 100 150 200 250 300 350 Time Since Stimulus (min) page 4 of 61 pages % Maximum Contents Release % Maximum Contents Release % Calcein Release 100 50 0 100 100 -20 80 60 40 20 0 -20 80 60 40 20 0 R 1 = Br- R 1 = CH 3 - R 1 = n-propyl-NH R 1 = H 0 50 100 150 200 250 300 2- time since S O addition (min) 2 4 10% 3.8% 0% [DOPE] 0 10 20 30 40 50 60 70 80 90 Time Since Stimulus (min) 0.050 M PB with Added 0.075 M KX Salt K SO 2 4 KCl PB Only KSCN Kosmotropic Chaotropic 0 20 40 60 80 100 120 140 160 180 200 220 240 Time Since Dithionite Addition (min) Figure 2. Top: Contents release at pH 7.4 for ~110-nm diameter (see cryoTEM image, inset) QR1-DOPE liposomes after chemical reduction at t=0 for various quinone head groups (see Table 1). Middle: Effect of added DOPE diluent lipid on contents release from PEG-PE/QMeMe-DOPE liposomes. Bottom: Hofmeister effects on contents release from QMeMe-DOPE liposomes. Green: Q Me Rho110 Blue: DRAQ5 (Nuclei) 25 μm 100 nm so as to probe for chemical targets within the endosomal process, as outlined in Figure 3 below. A549 (Positive) Figure 3. Quinone-cloaked Rhodamine imaging dyes. Left: Demonstration of fluorescence de-cloaking as a result of chemical reduction of QMeRho110 dye by dithionite. Middle: Chemical selectivity of dye de-cloaking for Q MeRho110 in the presence of possible interferents. Right: Confocal fluorescence microscopy image for A549 cancer cells (NQO1 positive) after incubation with QMeRho110 dye and nuclear staining dye DRAQ5.
Robin L. McCarley September 2011 For all of the stimuli-responsive soft matter systems, we foresee numerous target stimuli as possible avenues of investigation, such as amidases, esterases, and small-molecule biomarkers. Thus, the variety of possible materials we can make will have a significant impact on numerous application areas in nanomedicine, both from diagnostic and possible treatment avenues. Increasing the Functional Capabilities of Microfluidic Devices by Surface Chemical Modification and Creation of Nanostructures within Microstructures (NIH and NSF funding) Overview. Our group is a pioneer in the chemical modification of polymer surfaces that allow for attachment of key biological species, small molecules, and metallic features to the polymer surface, so as to augment the capabilities of microfluidic analysis devices made from them, Figure 4 (publications #37, 38, 43, 46, 48, and 49). Of late, we have targeted the creation and modification of ultra-high surface area polymer nanostructures that are integrated into the micrometer scale features of these devices, so as to yield highly responsive fluorescence-based arrays and capture surfaces, and highly efficient flow-through reactors (publications #72, 73, and 75). Surface Modification of Plastic-Based Microdevices Development of Surface Modification Protocols Embossed PMMA Electrophoresis Device Patterned Metal Deposition - Circuitry - E-chem Detectors - Conductivity Dets EOF(x10 -4 cm 2 EOF(x10 /Vs) -4 cm 2 /Vs) 4 2 0 Pristine COOH-Terminated -2 NH -Terminated 2 C -Terminated 18 2 4 6 8 pH 10 12 X X X X Plastic Surface Immobilization of Biomolecules - DNA Arrays - Protein Adhesion - Enzyme Rxns ChemModMachOver Figure 4. Chemical modifications of polymers and their applications for microanalytical devices resulting from McCarley Group research. species can be attached/grafted. These approaches have resulted in the successful development of microfluidic DNA arrays, electrical connections for device communication, rare cell-capture (circulating tumor cells) and release modules, and highly efficient solid-phase proteolytic reactors/processors. Current and Future Work. Our investigations are focused on the creation and use of surface modified devices in bioanalysis, particularly in rare event and parallel processing analyses that are important for detection strategies for a variety of diseases, including cancers, and for the screening of potential drug candidates. We have been working on the development of stimuli-responsive binding surfaces that allow for the reversible capture and release (concentration) of target analytes from flowing streams wherein there exist a myriad of similar biological species. In addition, we are The modified polymer surfaces of what are referred to as “micro-total analysis” or “lab-on-a-chip” systems play a significant role in the general utility of these miniature analytical measurement systems. Devices made from these polymeric materials offer the advantage that that they can be rapidly mass produced, often at a cost and with greater simplicity than glass or silicon-based devices, such that they have the potential to be used in a clinical setting. Prior to our work in this area, the surface chemical modification of the materials traditionally used to construct these devices— poly(methyl methacrylate), poly(carbonate), and cyclic olefin copolymer—was limited at best. We have designed and implemented a variety of solution, light-directed, and gas-phase methods for the chemical modification of polymer surfaces in microfluidic devices. For example, we have shown that roughly monolayerequivalent chemical functionalization of these three polymer surfaces can be achieved so as to yield both patterned (micrometer resolution) and non-patterned chemically reactive surfaces onto which an important selection of page 5 of 61 pages Response (RU) 100 0 biot-MBP Association Buffer Run biot-MBP Dissociation pH 10 Regeneration Buffer Run 0 200 400 600 800 1000 time (s) Figure 5. Representative surface plasmon resonance sensogram for regeneration of nitroavidin ligand surfaces by changing the pH of the system to basic conditions (pH 10 Na 2CO 3) for ~180 s at t=~600 s. Target analyte is biotinylated myelin basic protein, biot-MBP.
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